Microbial enhanced oil recovery methods

ABSTRACT

The present invention is directed to the field of microbial enhanced oil recovery (MEOR). In particular, the invention focuses on new, efficient, economical and environmentally safe microbial methods to enhance oil recovery in existing oil reservoirs, as well as microorganisms useful in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. Section 119(e) and thebenefit of U.S. Provisional Application Ser. No. 61/238,044 filed Aug.28, 2009, the entire disclosure of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention is from the field of microbial enhanced oilrecovery (MEOR). In particular, the invention concerns new, efficient,economical and environmentally safe microbial methods to enhance oilrecovery in existing oil reservoirs, as well as microorganisms useful insuch methods.

BACKGROUND OF THE INVENTION Background

The demand for crude oil has exceeded the existing production in theUnited States for more than 30 years. This has led to increasing demandfor more imported oil and a dependency on foreign suppliers. The growthof emerging economies is rapidly increasing the demand for oil in theglobal market. It has been estimated that more than half of allconventional oil (oil that can be produced with current technology) hasbeen produced. Most of the remaining conventional oil is located in theEastern Hemisphere or in environmentally sensitive areas such as theNorth Pole. The lack of conventional oil supplies could keep oil pricesso high that oil dependent nations such as the United States would beunable to fund the development of alternative energy technologies and beforced into dependency on foreign alternative energy as well. Thereforeany new technology that could increase the efficiency of oil recoverywould be of great benefit to countries such as the U.S. that have largeamounts of unrecoverable oil in place (OIP) in older exiting oil fields.

Most oil fields are small and are spread out in the 600 or so sedimentbasins throughout the world. Most of these oil-producing basins havebeen explored. Generally the largest fields are discovered first, andfurther exploration finds only smaller reservoirs. Most of the world'spetroleum is found in large fields. Only 37 supergiant oil fields ofover 5 billion barrels have been found. These 37 fields account for 80%of all the known oil. Only two of these supergiants are in North Americaand 26 are in the Persian Gulf Most of the remaining undeveloped oil inthe Western Hemisphere is not light petroleum, but is heavy oil or tarsands. Large deposits of heavy oil are in Venezuela and California.Canada has large deposits of tar sands. Currently, production of heavyoil requires large amounts of energy.

Most petroleum is found in sandstone, siltstone or carbonate. Porositiesvary from 5% to 30%. The porous rock, covered with an impermeable layer,collects oil from organic matter in lower source rock. It is a processthat takes millions of years. The maturation process converts it to acomplex mixture of hydrocarbons of about 82 to 87% carbon and 12 to 15%hydrogen. The oil moves into the porous rock in low concentrations withwater. To become a reservoir the porous rock must have some type ofimpermeable cap-rock that traps the oil. Most traps are anticlinalupfolds of strata that are oval shape, however, fault-traps andsalt-domes are also common. Oil near the surface often encountersdescending meteoric water that brings in oxygen and bacteria thatdegrade the oil to heavy oil or tar. Oil is usually not found below4,900 meters because the high temperature of deep rock will degrade thepetroleum into natural gas. Therefore, most oil is between 760 m and4,900 m deep.

Unlike natural gas, the recovery of petroleum oil is not efficient. Theexisting conventional oil production technologies are able to recoveronly about one-half of the oil originally in place in a reservoir oflight oil. For heavy oil, the recovery is often less than 10%. Tar sandsare so heavy that they will not flow at all and no oil can be recoveredby conventional drilling and pumping. A technology that could recover agreater percentage of this residual oil could increase oil productionfrom existing reservoirs and reduce the need of the U.S. to importedoil. The additional oil recovered from existing oil producing reservoirscould reduce the need to explore and develop wilderness areas that arepotential new oil fields. This additional recovery of existing oil couldbridge the gap needed for the development of alternative renewableenergy sources.

The Original Oil In Place (OOIP) is the petroleum present in the oilreservoir when first discovered. The volume of the reservoir isdetermined by the size and porosity of the carbonate or sand stone. Theporosity of the rock is a measure of the amount of small chambers ormicro-traps within the rock that can hold water or oil. The oil isgenerally pushed up to the surface with the existing oil reservoirpressures at first. The pressure in the oil well drops with time andthere is a need to create overpressure with other means such as waterinjection or a gas injection for secondary recovery of the OOIP. Thechoice of a specific secondary recovery technique depends on the type ofthe hydrocarbon accumulation and the nature of the reservoir. Waterinjection or “water sweep” or “waterflooding” is a common secondaryrecovery technique. In waterflooding, pressurized water is injected intothe oil-bearing formation rock. Ideally, the injected water displacesthe residual oil and moves it to a producing well. Generally inwaterflooding, crude oil free of water is recovered first, and thensubsequently a mixture of crude oil and water are recovered from theproduction wells. At some point, the percentage of water in theoil-water mixture (referred to as the water cut) from this techniquebecomes so high that it is uneconomical to continue pumping oil from thewell. The problem, with using water as a “drive fluid”, is that waterand oil are immiscible. The lower viscosity water will flow over the oiland by-pass large amounts of oil. Therefore, even after secondaryrecovery, a significant portion of crude oil remains in the formation,in some cases up to 75% of the OOIP. The fraction of unrecoverable crudeoil is typically highest for heavy oils, tar, and large complexhydrocarbons. In the U.S. this residual OIP in old oil wells could be asmuch as 300 billion barrels of light oil. World-wide, the estimate ofunrecoverable oil is 2 trillion barrels. There are an additional 5trillion barrels of heavy oil, most of which is unrecoverable. Much ofthis remaining oil is in micro-traps due to capillary forces or adsorbedonto mineral surfaces (irreducible oil saturation) as well as bypassedoil within the rock formation.

Enhanced Oil Recovery

Oil recovery by injection of fluids not normally found in the reservoiris referred to as Enhanced Oil Recovery (EOR). It is a subset ofImproved Oil Recovery (IOR), which can include operational strategiessuch as infill drilling and horizontal drilling. Although it issometimes referred to as tertiary recovery, it can be implemented alongwith secondary processes. Many types of EOR have been proposed and usedover the years. Technical complexity and the high cost of chemicals haveprevented the widespread use of EOR to where it only represents about10% of total United States oil production.

There have been two major EOR approaches; thermal and non-thermal.

Thermal Processes

Thermal processes work by heating the reservoir rock and the oil toreduce viscosity of the heavy oil. In general, the lower the viscosityof the oil, the better its recovery will be. The most widely usedthermal process is steam injection in which the temperature of thereservoir and the remaining oil is increased by heat energy of steam.Hot water may also be used, but it is not as efficient at transferringheat to the oil and rock in the reservoir. Unfortunately, in bothprocesses, most of the heat energy is lost to the surroundings and doesnot go to heating the oil. In situ combustion of the oil is much moreefficient than steam because it only heats the reservoir and not all thepipes and overburden rock. However, in situ combustion is difficult tocontrol and is seldom used. Typically, it requires the energy equivalentof a half a barrel of oil to recover a barrel of oil with a steaminjected thermal process. However, this depends on the oil saturationand the configuration of the reservoir. Because most of the energycarried by the steam is given up to the pipes, wall rock, and reservoir,it is best to use only on reservoirs with a high oil content so as torecover as much oil as possible with the steam used to heat thereservoir rock. Generally, thermal methods are used on heavy oil becauseit reduces the viscosity of the oil and increases the mobility of theoil and the mobility ratio (mobility of displacing fluid to mobility ofdisplaced fluid or oil). Typically, recoveries are in the range of 50 to60% for a thermal process, but the net energy gain is much less thanthat because of the large amount of energy needed to make steam.

Non-Thermal Processes

Several non-thermal processes have been experimented with or used overthe years. These rely on a combination of reducing the oil viscosity anddecreasing the interfacial tension (IFT) between the oil and displacingfluid. Ideally, the mobility of the displacing fluid should not behigher than the oil. The mobility ratio (mobility of displacing fluidover mobility of displaced fluid) should be low. The mobility of the oilcan be increased by viscosity reduction and by IFT reduction. As the IFTis decreased, the oil becomes more miscible with the fluid until itbecomes one phase and the IFT is zero. This decreases the mobility ratioand increases the oil recovery. Alternatively, the viscosity of thedisplacing fluid can be increased by adding polymers to “thicken” theliquid. Non-thermal methods require less energy and are best suited forlight oil of 100 cp or less. However, most non-thermal methods requireconsiderable laboratory experimentation and process optimization.

Microbial Enhanced Oil Recovery (MEOR)

One special type of EOR technique uses microorganisms such as bacteriaand archaea to dislodge the micro-trapped or adsorbed oil from the rock.The goal of this technique, which is known as microbial enhanced oilrecovery (MEOR), is to increase oil recovery of the original subsurfacehydrocarbons using bacteria rather than the more costly chemicalrecovery processes. These biological processes typically usemicroorganisms to achieve similar results as the chemical methods inthat they reduce IFT and reduce the mobility ratio of the water drivefluid to oil. The major mechanisms that microbes are believed to operateby are they: (1) alter the permeability of the subterranean formation byproducing low molecular weight acids from the biodegradation ofhydrocarbons which cause rock dissolution, (2) produce biosurfactantsthat can decrease IFT and form micelles of oil in water, (3) mediatechanges in wet-ability of the oil droplet by growing on the droplet andchanging the surface of the oil to a less hydrophobic surface (4)produce bio-polymers that improve the mobility ratio of water topetroleum by increasing the viscosity of water and plug high flowchannels, (5) produce lower molecular weight hydrocarbons byenzymatically cleaving the large hydrocarbons into smaller molecules,and thereby reduce the oil's viscosity, (6) generate gases(predominantly carbon dioxide and nitrogen) that increase formationpressure.

Of all the EOR processes, MEOR is presently considered the lowest costapproach, but is generally the least often used. The main reason thisbiological process is not more widely used, is that it is not alwayssuccessful or predicable. Furthermore, bacteria in oil wells, pipes andtanks are known to cause problems. In fact, it is believed that highviscosity heavy oil such as oil sands are the result of bacteriaconsuming the lighter weight petroleum components and leaving behind thehigh molecular weight fractions which are less readily consumed by thebacteria. Therefore many petroleum engineers see bacteria as a problem,not a solution. In fact, if not used correctly, the growth of bacteriacould degrade the oil or increase the hydrogen sulfide concentration inthe reservoir.

Numerous microorganisms have been proposed for achieving variousmicrobial objectives in subterranean formations. Early MEOR techniquesinvolved injection of an exogenous microbial population into old and lowproducing oil wells. The inoculating culture was supplied with nutrientsand mineral salts as additives to the water pumped into wells for oilrecovery. The development of exogenous microorganisms has been limitedby the conditions that prevail in the formation. Physical constraints,such as the small and variable formation pore sizes together with thehigh temperature, salinity and pressure of fluids in the formation andthe low concentration of oxygen in the formation waters severely limitthe types and number of microorganisms that can be injected and thrivein the formation. Later, it became apparent that indigenous microbesstimulated by the nutrients were playing the major role in oil recovery.Accordingly, many attempts at biological oil recovery do not injectbacteria at all, but rely on indigenous microorganisms exiting in theextreme environment of the oil reservoir.

Biological constraints, such as competition from indigenous microbes andthe stress of changing environments (from surface to subsurface) alsoact to limit the viability of exogenous microorganisms. To overcomethese problems, the use of indigenous microorganisms, commonlyanaerobic, has been proposed in MEOR projects. It is known that bacteriaand other microbes that can grow indigenously within petroleum oilreservoirs and can be used to enhance oil production. It is also knownthat bacteria and other microbes will metabolize various components ofpetroleum as a carbon and energy source. In addition to the beneficialeffects of making surfactants, solvents and other metabolites that canresult in an increase in oil production; they can also consume oil as acarbon source. Unfortunately, they especially prefer to consume theshort-length alkanes, not the heavy viscous oil.

In fact, the process of petroleum bio-degradation relies on theemulsification of oil so that the hydrocarbon can be transported intothe bacterial cells for conversion to fatty acids as a carbon and energysource. This process can be used to remediate oil spills and other oilcontaminated sites by supplying the indigenous microbes with nutrientsor inoculating with cultures of microbes that can degrade oil. In thecase of biological remediation of petroleum contaminated sites, microbescan produce metabolites such as surfactants that help emulsify oil sothat they can then use the emulsified oil as a carbon source. Theprocess of petroleum bio-degradation relies on the emulsification of oilso that the hydrocarbon can be transported into the cell for conversionto fatty acids as a carbon and energy source. Both of these functionshelp remove the hydrocarbon contamination from the site. However, in thecase of MEOR only the production of metabolites such as surfactants,bio-polymers, hydrocarbon cleaving enzymes, organic acids and solventsare beneficial to increased oil production. Other than providing anenergy source, the consumption of light petroleum is not beneficial toenhanced oil production from the reservoir.

The biodegradation of the shorter carbon alkane chains reduces thelighter fraction of the hydrocarbon mixture in the petroleum oil. Theremoval of the short chain alkanes from this mixture increases theoverall viscosity of the hydrocarbon mixture. The higher viscosity ismore difficult to recover from the reservoir. The percent of recoverableoil is decreased. Also the oil that is recovered is more difficult totransport through pipes and to refine. Therefore the production ofuseful compounds, by microbes for improved oil recovery, comes with ahigh cost.

The process of stimulating all the indigenous microbes in an oilreservoir by adding nutrients is therefore unpredictable. The growth ofthe microbes could produce the beneficial effect of dislodging oilentrapped within a petroleum reservoir. Alternatively, the side effectof light oil consumption could make the oil more viscous and lower thetotal recovery of oil.

It would be less detrimental if all petroleum components were degradedequally, but the case is that the shorter chain alkanes and lowermolecular weight aromatics are more readily degraded by the microbes asa carbon and energy source. This is supported by the fact that petroleumdeposits near the surface and most subject to biodegradation aregenerally very high in high viscosity oil made up of high levels ofasphaltic hydrocarbon and fairly low on light (short) chain alkanes.Canadian tar sands are believed to be the heavy residue representingabout 10% of the petroleum deposit that has been degraded.

In the past, others have taught ways of augmenting the growth ofmicrobes that dislodge and mobilize oil from underground petroleumreservoirs. These methods generally recommend adding nutrients. Somehave also taught adding various cultures of selected bacteria that addedbeneficial capabilities. Some have even reported isolating microbes thatcan only degrade higher molecular weight hydrocarbons (U.S. Pat. No.5,013,654). However, adding these selected cultures is not enough.Although these prior methods disclosed that microbes exist that can onlyfeed on high molecular weight oil, they failed to provide methods ofincreasing the bio-digestion of heavy oils, while suppressing thelighter weight hydrocarbon consumption by other indigenous microbes. Themicrobes that were simply residing within the petroleum reservoir arelikely to have the ability to degrade lower weight oil. Adding needednutrients would stimulate the growth of all the microbes present.Because the smaller hydrocarbons can be transported across the cellmembrane, the light weight oil consumers will grow faster than the highweight oil consumers and predominate in the population that results fromstimulation.

The prior art does not teach methods that prevent the fasterbiodegradation of the light weight low-viscosity oil in comparison tothe slower biodegradation of the higher weight viscous oil. Therefore,the same process that is beneficial to oil recovery is also detrimentalto oil viscosity; and it is known that increasing the viscosity of theresidual petroleum held within the reservoir will decrease oil recovery.Therefore, prior methods of adding nutrients, either with or withoutspecially selected or engineered microbes, are unpredictable in terms oftheir ability to increase oil production.

Accordingly, there is a great need for new enhanced oil recoveryapproaches that are energy efficient, and can be reliably andsuccessfully used in large field situations to enable the recovery ofcurrently unrecoverable oil in existing oil fields.

SUMMARY OF THE INVENTION

It is an object of this invention to provide microorganisms with genesthat are useful to enhance recovery of petroleum oil from undergroundreservoirs, oil sands and other sources of heavy oil while suppressingthe consumption of the lighter fraction of the petroleum. In addition,it is an object of this invention to give the host or recipient organismof these genes a competitive advantage for the special environment ofthe hydrocarbon resource reservoir.

It is another object of the present invention to enable the recovery ofoil in existing oil fields or other oil reservoirs where such recoverywould otherwise not be commercially feasible.

In one aspect, the present invention concerns a method of enhancing oilrecovery comprising introducing into an oil reservoir a microorganism,which is a halophile and is deficient in its ability to degrade shortchain hydrocarbons of about 12 carbons or less. Preferably, the growthof such microorganism is obligately dependent on high salinity, i.e. itis an obligatory halophile.

In one embodiment, the microorganism is of the domain Archaea(hereinafter referred to as archaea) or is a bacterium.

In another embodiment, the microorganism, such as an archaea or abacterium, is present in a culture of microorganisms or in a consortium,while in another embodiment, it may be able to grow in a salinity ofabout 50,000 ppm or higher.

In a further embodiment, the microorganism is inhibited in acquiring theability to grow at salinity below about 50,000 ppm from microorganismsindigenous or contaminating the reservoir.

In yet another embodiment, the microorganism is deficient in its abilityto degrade hydrocarbons of about 20 carbons or less.

In a further embodiment, in the microorganism one or more metabolicpathways degrading short chain hydrocarbons of about 12 carbons or lessare down regulated or deleted.

In a still further embodiment, the microorganism naturally lacks theability to degrade short chain hydrocarbons of about 12 carbons or less.

In a different embodiment, the microorganism has the ability to utilizearomatic hydrocarbons.

In another embodiment, the microorganism has the ability to utilizehydrocarbon chains of greater than about 12 carbons, or about 20hydrocarbons.

In yet another embodiment, the microorganism has the ability to utilizemodified hydrocarbons containing sulfur.

In a further embodiment, the microorganism has the ability to utilizemodified hydrocarbons containing nitrogen.

In a still further embodiment, the microorganism has the ability toutilize simple carbons from the group comprising; glucose, sucrose,mannose, starch, glycerin, organic acids, and other simple sugars.

In a different embodiment, the microorganism has the ability to producesurfactants.

In another embodiment, the microorganism has the ability to produceextra cellular polymers.

In a further embodiment, the microorganism (i) contains functional genesfor the metabolism of high molecular weight hydrocarbons; (ii) lacksfunctional genes for the transport and/or oxidation of short chainalkanes at the cell membrane; (iii) contains functional gene or genesfor the production of surfactants; and (iv) is regulated to express saidfunctional gene(s) and grow in a high salt environment within areservoir.

In all embodiments, the method may further comprise the step ofwater-flooding the reservoir with low salinity fluid or a fluidcontaining a compound toxic to said obligatory halophiles to reduce theconcentration of halophilic microbes that have the ability to utilizeshort chain hydrocarbons of about 12 carbons or less, or 20 carbons orless.

In all embodiments, the method may further comprise the step ofinjecting a nutrient mixture into the reservoir.

In another aspect, the invention concerns a microorganism that (i) is ahalophile, and (ii) is deficient in its ability to degrade short chainhydrocarbons of about 12 carbons or less.

In one embodiment, the microorganism is an obligatory helophile.

In another embodiment, the microorganism is an archaeon or a bacterium.

In another aspect, the invention concerns a microorganism of the domainArchaea or bacteria that (i) is an obligatory halophile, (ii) isdeficient in its ability to degrade short chain hydrocarbons of about 12carbons or less, and wherein (iii) the growth of said microorganism isobligately dependent of high salinity, wherein in various embodiments,the microorganism naturally has and/or is engineered to have theproperties (i)-(iii).

In yet another embodiment, the microorganism is able to grow in asalinity of about 50,000 ppm or higher.

In a further embodiment, the microorganism is inhibited in the abilityto acquire the ability to grow at salinity below about 50,000 ppm frommicroorganisms indigenous in or contaminating an oil reservoir.

In a still further embodiment, the microorganism is deficient in itsability to degrade hydrocarbons of about 20 carbons or less.

In a different embodiment, the microorganism has the ability to utilizearomatic hydrocarbons.

In another embodiment, the microorganism has the ability to utilizehydrocarbon chains of greater than 12 carbons.

In yet another embodiment, the microorganism has the ability to utilizemodified hydrocarbons containing sulfur.

In a further embodiment, the microorganism has the ability to utilizemodified hydrocarbons containing nitrogen.

In a still further embodiment, the microorganism has the ability toutilize simple carbons from the group comprising; glucose, sucrose,mannose, starch, glycerin, organic acids, and other simple sugars.

In a different embodiment, the microorganism of has the ability toproduce surfactants.

In another embodiment, the microorganism has the ability to produceextra cellular polymers.

In another aspect, the invention concerns a culture of consortiumcomprising a microorganism as hereinabove described. In variousembodiments, the culture or consortium may comprise, consist essentiallyof or consist of a plurality of microorganisms as hereinabove defined.In addition, a consortium may comprise microorganisms of different type(e.g. both bacteria and archaea) and/or having different propertiesselected from the characteristics described above and/or otherwisedisclosed herein.

It is noted that two or more of the various embodiments listed above orotherwise disclosed herein can be used in any combination, and any andall of such combinations are within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process of replica plating that can be used as partof the present invention.

FIG. 2 illustrates the osmotic adaptation of enzymes.

FIG. 3 is a photograph of a gel showing the expression of the ladA genein E. coli and in Haloferax volcanii. NuPAGE 4-12% Bis-Tris gel(Invitrogen, Cat # NP0323BOX) analysis of Ni-NTA column purified nativeLadA (no amino acid residue change) from E. coli BL21(DE3) (GFF40) andmut3 LadA (with 3 amino acids changed) from Haloferax volcanii (GFF31);

1: GFF40 cell lysate (CL) prior IPTG induction;

2 & 4: GFF40 CL 3 hours post IPTG induction;

3: SeeBlue Plus 2 protein standard;

5: GFF40 CL flow through (FT) Ni-NTA column;

6: 10× volume Ni-NTA column wash solution passed the Column;

7: elute from GFF40 column using elution buffer;

9: elute from GFF31 column

10: elute from HVev [Haloferax vocanii (HV) containing an empty vectorpng168] column

11: elute from HV column.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further the actualpublication dates may be different from those shown and requireindependent verification.

The term “oil reservoir” is used herein in the broadest sense andincludes all forms of hydrocarbon deposits, including, withoutlimitation, underground reservoirs, producing wells, non-producingwells, experimental wells, exploratory wells, oil sands and othersources of heavy oil and the like, which may be accessible by any means,such as, for example, one or more wellbores.

The terms “microorganism” and “microbe” are used interchangeably and inthe broadest sense, including all types of microorganisms, includingbacteria, fungi, archaea, and protists, and microscopic animals, such asplankton, planarian and amoeba. Preferred microbes for the purpose ofthe present invention are bacteria and archaea.

The term “microbial consortium” is used herein to refer to multipleinteracting microbial populations. Members of a consortium communicatewith one another. Whether by trading metabolites or by exchangingdedicated molecular signals, each population or individual detects andresponds to the presence of others in the consortium. This communicationenables a division of labor within the consortium. The overall output ofthe consortium rests on a combination of tasks performed by constituentindividuals or sub-populations.

Archaea comprise one of the three distinct domains of life, withbacteria and eukaryotes. For a review, see, e.g. Makarove and Koonin,Genome Biology 4:115 (2003).

The term “halophile” is used herein to refer to an extremophile thatthrives in environments with very high concentrations, typically atleast about 5% (50,000 ppm), or at least about 10%, or at least about15% of salt.

The term “obligatory halophile” is used herein to refer to anextremophile whose growth is obligately dependent on high saltconcentrations, typically at least about 5% (50,000 ppm), or at leastabout 10%, or at least about 15% of salt.

The terms “repression” and “inhibition” with reference to geneexpression are used herein interchangeably and refer to any processwhich results in a decrease in production of a gene product, regardlessof the underlying mechanism. A gene product can be either RNA orprotein. Gene repression includes processes which decrease transcriptionof a gene and/or translation of mRNA. Thus, specifically included inthis definition are processes that inhibit the formation of atranscription initiation complex along with those that decreasetranscription rates and those that antagonize transcriptional activationis gene repression. These repressions can be either reversible orirreversible, both of which are specifically included in thisdefinition.

The term “lateral gene transfer” is used herein in the broadest senseand refers to the transmission of genetic information from one genome toanother.

The term “surfactant” as used herein means microbially producedsurface-active agents, including, but not limited to, glycolipids (e.g.sophorose lipid or rhamnose lipid), lipoproteins, polysaccharide-fattyacid complexes, mono- and diglycerides, lipoheteropolysaccharides,peptidolipids, neutral lipids, corynomycolic acids, trehalosedimycolates and polysaccharide-protein complexes.

The term “hydrocarbon” is used herein in the broadest sense to describeany organic compound that contains only carbon and hydrogen. The termspecifically includes, without limitation, straight and branched chainedsaturated hydrocarbons (alkanes), straight and branched chainedunsaturated hydrocarbons (including alkenes and alkynes), cycloalkanes,and aromatic hydrocarbons (arenes).

A “short chained alkane”, as defined herein, contains 1 to 4 carbonatoms.

A “high molecular weight hydrocarbon”, as defined herein, is ahydrocarbon having at least about 40 carbons, for example, a hydrocarbonhaving between about 40 and about 60, or between about 40 and about 80,or between about 40 and about 100, or between about 40 and about 120carbons.

II. Detailed Description

In the present invention, means are provided to maintain the beneficialaspects of oil mobilizing microbes, while preventing their detrimentaleffects. One major detrimental effect of microbes is that they can growin a petroleum reservoir and consume petroleum. Not only do microbesconsume petroleum, but they are much faster at consuming the lighterfraction of petroleum oil. The methods provided by this invention aredesigned to stop or reduce the consumption of beneficial light weightpetroleum by the consortium of microbes that is used to make surfactantsand other metabolites that are beneficial to enhanced oil production.

According to the present invention, the detrimental effect of light oilconsumption is selected out of the consortium. Bacteria and othermicrobes are carefully selected or modified to be deficient in theirability to consume the lower weight hydrocarbons, especially the shorterchain length (e.g. less than about 20 carbons, or about 12 carbons orless) alkanes. The preferred consortium of microbes is selected,modified or controlled so that the microbial culture relies on either asoluble carbon source provided by the nutrient mixture injected with themicrobes and water flood or the bio-consumption of high molecular weighthydrocarbons present in the petroleum. The metabolic pathways thatdegrade only the higher molecular weight hydrocarbons are notdeliberately down regulated or deleted. These pathways are beneficialbecause not only does the consumption of higher molecular weightcarbohydrates provide an additional carbon source, but the removal ofhigh molecular weight oil reduces the viscosity and improves both thevalue and the recovery of the petroleum.

However, the selection or engineering of a microbial culture that lacksthe detrimental effects of consuming light oil is not sufficient. Such aculture could be quickly overcome by indigenous or contaminatingmicrobes that had the ability to consume light weight oil. Even if theengineered culture was robust and quick growing it could acquire genesthat coded for the metabolism of light weight oil. This could occur bythe process of Lateral Gene Transfer (LGT), which is known to occur inmany natural environments. Picking up such genes would give the microbesa competitive or evolutionary advantage and they would soon dominate thepopulation.

This invention provides a means for preventing LGT (also known ashorizontal gene transfer) so that the engineered or selected culture ofmicrobes does not acquire unwanted or detrimental pathways for improvedoil production. For example, a bacterium that only had genes coding forthe enzymes that were required to metabolize polyaromatics, but did nothave the genes coding for the enzymes needed for short chain alkaneutilization would be good for oil recovery. If it acquired genes fromanother bacterium that also gave it the ability to metabolize shortchain alkanes it would grow faster and would increase in population.This would be a beneficial adaption for the bacteria, but would notbenefit the oil recovery process, since it could increase the viscosityof the remaining petroleum mixture

Lateral gene transfer is common among bacteria and Archaea as amechanism of genetic information sharing between different species. Itis believed to play a significant role in evolution and is also known tooccur in higher organisms. It is somewhat analogous to a computerprogram being transferred from one computer to another. A gene thatcodes for specific enzyme can be transferred from a bacterium to yeastin such a way that the yeast could produce the enzyme and obtain thatactivity. This process can occur in nature as well as in the laboratory.

Some Archaea and bacteria that live in very high salt environmentsmaintain very high salt concentrations inside the cytoplasm. Thisrequires major changes to the surface charges on proteins, enzymes andother cytoplasmic compounds so that they can remain soluble and functionin the high salt, low water environment. The process that brought aboutthese changes to “salt in” halophiles, especially obligate halophiles,is believed to have taken thousands, if not millions of years to evolve.Therefore, a gene transferred into a halophile from a non-halophile orlow salt cytoplasmic microbes is unlikely to be functional or produce afunctional gene product in a “salt-in” halophile. This invention relieson this different high salt “operating system” of obligate halophilicArchaea and bacteria to prevent LGT of short chain utilizing enzymesfrom non-obligate microbes being acquired by the selected or engineeredhalophiles for MEOR.

With the ability to prevent the metabolism of light oil, thecorresponding viscosity increase, cased by the removal of the light oilfractions, is also prevented. As stated above, the higher the viscosityof oil, the lower the recovery will be. The beneficial effects of themicrobes such as reduction of IFT, increase sweep efficiency andimproved mobility ratio could be negated by increase in viscosity.However, if the microbes can only consume heavy oil or other carbonsources the major detrimental effect can be avoided. This makes the MEORprocess of the present invention more predictable and more effective.

The use of an obligate halophile requires that any new genes taken fromnon-halophiles or low salt-in cytoplasm (non-obligate) halophiles bemodified to change the amino acid residue sequences of any proteins theycode for. These changes are needed to render the proteins functional andare more soluble at high salt concentrations. These required changes canbe determined by analysis of homologues proteins found in bothnon-halophiles and obligate halophiles. In addition, three dimensionalstructural analyses can be used to determine surface positively chargedresides such as lysine which may be changed to negatively charged aminoacid residues such as aspartic acid. This type of amino acid residuechanges will result in an increase negative charge, which is generallybeneficial to high salt functionality. A large number of potentiallybeneficial changes can be made and then tested by expression in a modelobligate halophile such as Haloferax volcanii.

The study of hydrocarbon bio-degradation provides an understanding ofthe mechanism of short chain alkane metabolism. The shorter chainalkanes are made soluble in water generally with the aid of surfactantsproduced by the bacteria or Archaea. Then the soluble alkane adsorbsonto the cell's hydrophobic membrane and is transported across themembrane of the microbe. Enzymes bound to the membrane convert thealkane to an alcohol. Subsequent chemical reactions catalyzed by otherenzymes convert the alcohol to an aldehyde and then to an organic acidalso referred to as a fatty acid. The fatty acid can then be furthermetabolized by the cell for energy and carbon building blocks for itsgrowth. This biology of the short chain alkane metabolism is the moststudied and the best understood. Metabolism of larger or highermolecular weight hydrocarbon is more complex and less well understood.The larger or higher weight hydrocarbons are much less soluble and moredifficult to transfer across the cell membrane. However, thebiodegradation of high molecular weight oil is known to occur, however,it happens at a slower rate. A more detailed description of biologicaldegradation of hydrocarbons is reviewed by J. D. Van Hamme, A. Singh,and O. Ward in Microbiology and Molecular Biology Reviews, December2003, p. 503-549. This invention relies on retarding the light chainalkane metabolism by the engineered or selected microbes.

In the methods of the present invention, genes that code for alkanehydroxylase systems which are capable of degrading light weight and lowviscosity hydrocarbons, are inhibited, e.g., deleted, mutated or downregulated in the selected or engineered microbe. In addition, LGT fromthe environment is prevented. That is, the acquisition of similar genesthat code for degradation of light weight oil from other microbes thatare present or could contaminate the reservoir is prevented. However,the production of surfactants or other metabolites, beneficial to oilmobilization, is not prevented. The expression of genes needed for theproduction of surfactants is maintained without the consumption of lowviscosity oil.

Different sets of genes code for each of the various metabolic functionsthat make possible hydrocarbon dependent microbes. The degradation andconsumption of the higher molecular weight hydrocarbons is generallyenabled by different genes from those in the light chain metabolicpathways. Genes that code for production of important degrading enzymesof hydrocarbon and genes for surfactant production may be regulated bythe same promoters, but the gene and gene products are separate and canbe manipulated so that they can be independently controlled.

Enzymes that degrade hydrocarbons have different substratespecificities. The first step in the degradation of alkanes is theoxidation of either the terminal carbon or the second to the terminalcarbon to form a primary or secondary alcohol. The monooxygenases thatcatalyze the first step in the metabolism of hydrocarbons have bindingsites that show a preference or specificity for different lengths ofstraight chain alkanes. In addition there are monooxygenases that willoxidize aromatic hydrocarbons of different sizes. Many of the genes havebeen isolated and their sequences characterized. Many others have notyet been isolated, but are expected to have similar sequences anddifferent specificities. With probes based on highly conserved sequencesof key enzymes and variable binding sequence motifs that determinesubstrate specificity, new genetic information can be obtained frommicrobes inhabiting petroleum sites.

Currently, there is enough nucleotide and amino acid sequenceinformation available on monooxygenases required for the degradation ofvarious size and types of petroleum hydrocarbons that highly conservedregions have been identified. Some of these highly conserved sequencesare required for catalytic activity. Others are substrate specific andwill vary with the size and type of hydrocarbon that they oxidize. Forexample, the 8 histidine amino acid residues that are required forcatalytic activity in all alkane monooxygenases are in three histidineboxes (Hist1, HE[L/M]XHK; Hist2, EHXXGHH; and Hist3, LQRH[S/A]DHHA)reported by J. B. van Beilen et al in Applied and EnvironmentalMicrobiology, December 2002, p. 5933-5942. This knowledge can be used tosearch for microbes within an environment that can degrade varioushydrocarbons. Probes to the highly conserved sites can be used isolategenes that code for monooxygenases that exist within the microbesinhabiting petroleum rich sites. Further analysis based on sequences ofthe substrate specific sites can identify genes that code for highermolecular weight hydrocarbon utilization.

Although slower than light chain utilization, degradation of heavy chainhydrocarbons can provide a supplemental carbon source without detrimentto the value of the petroleum oil. Microbes have been isolated that canonly grow on heavy petroleum components. These have been shown to havegenes that code for enzymes that are specific for certain of the heavierhydrocarbons and lack the genes for utilization of the lighter shortchain alkanes. For example, L. Wang et al reported isolating Geobacillusthermodenitrificans NG80-2 from a deep subterranean oil reservoir innorthern China that degrades and metabolizes only long chain (C15-C36)n-alkanes, but not short-chain (C8-C14) n-alkanes. The complete genomesequence of G. thermodenitrificans NG80-2 has been deposited in theGenBank database (NC_(—)009328 and NC_(—)009329, GenBank CP000557) andis incorporated in the corresponding publication in Proc Natl Acad SciUSA, Mar. 27, 2007 p. 5602-5607 by reference. Comparison of gene codingfor protein sequences can be used to find specific substrate sequencesto make probes for either short or long chain alkane monooxygenases toscreen DNA isolated from a specific site or oil reservoir. By thismethod and other methods of microbiology, the microbes responsibledegradation of heavy and light oil in a reservoir can be identified.

In addition to high molecular weight hydrocarbons, petroleum oilcontains compounds that are not desirable to have in oil that will berefined into various petroleum products. One major group of undesirablecompounds is modified hydrocarbons high in sulfur. Sulfur can be thethird most abundant element in crude oil and is especially high in heavyoil. Lowering the sulfur content would increase the value of the crudeoil. Bacteria that are capable of selectively attacking the C—S bondshave been isolated and their metabolic pathways elucidated. Most strainsstudied have been aerobically grown and include; Rhodococcuserythropolis, Nocardia spp., Agrobacterium sp. Strain MC501,Mycobacterium spp., Gordona sp. Strain CYKS1, Klebsiella spp.,Xanthomonas spp., and the thermophile Paenibacillus. These bacteria havebeen shown effective at desulfurization of various sulfur containinghydrocarbons found in crude oil. However, the process is a two phase oiland water system that requires surfactants and energy-intensive mixing.To achieve sulfur removal rate of over 50% high water to oil ratios wereneeded in well mixed and aerated reactors. The critical aspects of theprocess include reactor design, product recovery and oil-waterseparation.

Another group of undesirable hydrocarbons are nitrogenous compounds.Crude oil can contain about 0.5% to 2.1% nitrogen with 70% or more aspyrroles, indoles and carbazole nonbasic compounds. These compounds arepoisons to cracking catalysts, toxic and result in air pollution.Removal of the nitrogenous compounds would increase the value of oilrecovered by the MEOR process. Several species of bacteria have beenisolated that contain metabolic pathways for the oxidativetransformation of nitrogenous compounds found in crude oil. A review ofthese bacterial processes was published by Kaiser, J. P. et al inMicrobiol. Rev. 60:483-498. The genes responsible for carbazoledegradation by Pseudomonas sp. strain CA10 were identified and clonedinto E. coli by Sato et al and were reported to transform a wide rangeof aromatic compounds. The results are published in J. Bacteriol. 179:4841-4849 in 1997.

Following modification of genes coding for these desulfurizationpathways or denitrogenation pathways to be functional and producefunctional proteins in the high salt cytoplasm of the host halophile,their incorporation into a culture designed for oil recovery can alsoreduce the sulfur or the nitrogen content of the recovered oil. However,because these are oxidative processes it is important that genesresponsible for light chain metabolism be eliminated so that the shortchain alkanes are not degraded.

The soluble carbon sources comprise simple sugars, glycerin, starch,fatty acids and other organic molecules that can be metabolized by theconsortium of microbes without relying on the same metabolic pathwaysencoded by genes for the metabolism of alkanes of about 20 carbons orless, such as about 12 carbons or less. If the host or recipientmicrobe, engineered for the oil reservoir environment, does not containadequate pathways for the utilization of inexpensive soluble carbonsources, genes required for those pathways could be transferred into thehost microbe. Given the opportunity to use a simple carbon source,hydrocarbon degrading microbes will often down regulate the geneclusters containing both the genes for alkane moonooxygenases, and thegenes for surfactants that aid in the uptake of hydrocarbons.

That is, by providing a soluble carbon and energy source at sufficientlevels to maintain living cells and cell growth, the indigenous microbesmay become nondependent on alkane hydrocarbon metabolism for growth andsurvival. This could lead to down-regulation and low expression of genesor even loss of the genes that code for enzymes that make usefulmetabolites such as surfactants that emulsify the insolublehydrocarbons.

Therefore, a means for maintaining high expression and levels of certaingenes must be provided. This can be done by number of molecular biologytechniques, including, but not limited to, placing the genes coding foreach of the metabolic products such as surfactant production under thecontrol of an inducible or constitutive promoter. This will allow forhigh expression by both transcription and translation of these genes.This is capable of preventing a down regulation that can occur with thewild type promoter when the cell detects a high level of easier tometabolize or preferred carbon source. In conventional MEOR processesthat use only naturally occurring cultures of oil consuming microbes incombination with indigenous microbes present in the oil reservoir, theaddition of too much of a simple carbon source, such as molasses, couldlead to a reduction of surfactant production and unexpectedly lower oilemulsification.

The problem with relying on naturally occurring microbial processes isthat they become less effective at both oil degrading and oil recoverywhen they are supplied with an easily metabolized carbon and energysource, such as molasses. However, not supplying any simple carbonsource could slow growth and also lead to low oil production. Inaddition, the lack of a supplied carbon source will select for thestrains of microbes that can utilize the hydrocarbons that exist withinthe oil reservoir. Furthermore, microbes that have genes that enablethem to consume light weight oil will grow and multiply faster than anymicrobe, added or indigenous, that only contains genes for heavy oilconsumption. Therefore, it is best to provide adequate carbon sourcesfor the engineered or selected strains so that they can grow fast enoughto prevail over the indigenous strains that have the ability tometabolize short chain alkanes.

Gene promoters contain specific DNA sequences and response elements thatare recognized by proteins known as transcription factors. These factorsbind to the promoter sequences recruiting RNA polymerase, the enzymethat copies or transcribes the gene coded for in the DNA into amessenger RNA (mRNA). The mRNA can then migrate to a ribosome where itis translated into a protein or gene product. The protein may be aproduct itself or it may be an important part of the metabolic pathwaythat is being controlled.

Gene repression and inhibition of expression refer to any process whichresults in a decrease in production of a gene product, whether by areversible or an irreversible process. A gene product can be either RNAor protein. Gene repression includes processes which decreasetranscription of a gene and/or translation of mRNA. For example, aprocess that inhibits the formation of a transcription initiationcomplex or those that decrease transcription rates or those thatantagonize transcriptional activation is gene repression.

An inducible promoter is one that is controlled or regulated by someextracellular factor that can increase or decrease the transcription andtranslation of genes into their products. In a specific example ofn-alkane degradation, the alk genes of Pseudomonas oleovorans areresponsible for the degradation of n-alkanes. These genes are located intwo gene clusters that are controlled by a promoter which is controlledby the AlkS protein. This protein is responsive to the hydrocarbonoctane. The presence of octane will increase or activate the expressionof these genes and their protein products. However, this same promoteris also down regulated or repressed by the presence of a preferredcarbon source such as organic acids. These bacteria would both emulsifyand degrade n-alkanes unless high levels of a preferred carbon sourceare supplied. In this case, the genes for hydrocarbon degradation wouldbe turned off. This would limit its usefulness to remediatinghydrocarbon contaminated sites and could become less effective if givenmore easily metabolized carbon sources. However, by inactivating thedown regulation of the promoter by preferred carbon sources, andinactivation of genes from the cluster that are needed for themetabolism of alkanes, this microbe can be engineered into an oilemulsifying bacterium that can grow on soluble carbon sources.

One means for inactivating the down regulation by the simple solublecarbon source is to mutate the sequence of the AlkS protein that bindsthe carbon source in such a way as to not affect the octane bindingsite. Another method is to transfer the genes coding for the surfactantor bio-polymer production pathway to be under the control of differentpromoter. This provides a way of controlling the production ofsurfactant or bio-polymer independent of carbon source.

By maintaining growth on a medium containing a soluble carbon source,the genes that code for short chain alkane hydrocarbon metabolism can byinactivated by a number of means. Methods suitable for inactivation ofthese genes include, but are not limited to, chemical mutagens and UVand other forms of radiation. In addition, functional genes can bereplaced by nonfunctional genes. The technology of gene silencer,developed by A. Fire et al., Nature 391(6669):806-11 (1998), has lead toa better understanding of how genes regulate mammalian cell function.These methods of inactivating specific genes can be used to locate keygenes responsible for any metabolic process that a cell or microbe cancarry out. In addition, if selected microbes for improved oil do nothave completed genomic sequences available in the public domain, theentire genome can be sequenced rapidly by current technology at a fairlylow cost.

One functional gene may be used to replace another functional gene. Thenew gene may also include a reporter gene for easy selection of microbescontaining the new gene. For example, a functional cluster of genes thatcode for a high molecular weight hydrocarbon metabolism pathway may beinserted into a host cell. It may replace a gene cluster for lowermolecular weight hydrocarbon metabolism pathway that has been remover orinactivated. In addition, the cell may be given a resistance gene for anantibiotic or other toxin. This is a commonly used method for selectingcells that have successfully incorporated new genetic material. Theselected cells can then be grown to large numbers various large scalefermentation techniques known to those skilled in the art biotechnology.

However, there is a potential problem with removing the short chainalkane metabolism genes. The genes that encode for the metabolism oflight chain hydrocarbons maybe in clusters with the genes that arerequired for the production of useful metabolites such as surfactantsfor the emulsification of oil. Because surfactants are secreted to helpthe transfer of short chain alkane across the cell membrane they may becombined with the alkane metabolizing genes or controlled by the samepromoter. In that case the up regulation of useful metabolites and thedown regulation of light chain metabolism may require more complex genemanipulation. That is, key enzymes for the metabolism of short chainalkane should be inactivated not the entire gene cluster related toalkane consumption.

One problem that can prevent the success of this approach is that thegenes that code for hydrocarbon emulsification of oil, which helps oilrecovery, add no benefit to the microbe if the oil is not being consumedby the bacteria. Also if the bacterial culture has a preferred carbonsource in the waterflood fluid, the genes for surfactant productionwould quickly be lost. A microbial population will generally only carrythose genes that are necessary for it to prosper in an environment. Ifthese genes are not needed, they are soon lost. This is why thenutrients, and especially the carbon source, must be carefullycontrolled if the process only depends upon wild type microbes to eitherclean up oil spills or recover oil from old wells. Therefore someadvantage must be given the engineered microbes to make them better ableto survive in the oil reservoir environment. The engineered microbesthat can only metabolize high molecular weight oil or produce oilemulsifying surfactants must have a competitive advantage overindigenous microbes that can metabolize short chain alkanes.

It is the object of this invention to provide microbes with genes thatare useful for the enhanced recovery of petroleum oil from undergroundreservoirs, oil sands and other sources of heavy oil while suppressingthe consumption of the lighter fraction of the petroleum. In addition,it is the object of this invention to give the host or recipientorganism of these genes a competitive advantage for the specialenvironment of the hydrocarbon resource reservoir. By means known tothose skilled in the art of molecular biology, genes that are isolatedfrom bacteria and Achaea that are indigenous to oil reservoirs ornaturally occurring oil seeps that provide beneficial mechanisms forenhanced oil recovery are transfused and expressed at high levels inhost microbes. The host microbes are chosen for their survival in theextreme environment of an oil reservoir. The host microbes are providedwith a selective advantage for the reservoir environment. In the presentinvention and in a specific case the selective advantage is high saltconcentration tolerance. In addition the engineered or modified microbescould have the ability to utilize a special energy and or carbon sourcethat is supplied in the waterflood fluid. Genes that code forconsumption of heavy oil or toxic petroleum components would also bebeneficial to both the microbe and oil recovery process. Thesebeneficial genes would be preserved or transferred into the constructedmicrobes.

This technology is implemented by inoculating an oil reservoir with aculture of one or more microbes each containing combinations of genesfor the various mechanisms that are beneficial for improved oilproduction. The methods of the present invention allow for a widevariety of designs, and thus a combination of mechanisms may be designedfor a particular type of reservoir. In addition, a means for controllingand maintaining high expression of these genes may be provided. Incertain embodiments, along with the microbes, the present invention alsoprovides the chemical component to create the right environment for themicrobes that also suppresses the indigenous microbes that might consumethe mobilized oil, especially the short chain alkanes. In this example,a high salt requiring culture of microbes, are inoculated in a saltwater fluid supplied to the oil reservoir. This increases the level ofsalt in the reservoir so as to be toxic to the indigenous microbes, butis preferred for the culture of inoculating engineered or selectedmicrobes. In that case, the indigenous organisms, which might consumelight weight oil or produce hydrogen sulfide, will be inhibited orkilled. Therefore, the added nutrients will benefit only the growth ofthe processed-designed microbes and not the growth of detrimentalindigenous microbes.

(1) Isolation and Selection of Oil Recovery Genes that Code for Proteinsand Pathways for MEOR

Over 100 oil degrading microbes have been isolated and reported. Manyhave been well studied and the sequences of genes related to variousfunctions of the petroleum oil degradation process published. In somecases, for example Alcanivorax borkumensis SK2, the complete genome of3,120,143 base pairs (bp) has been sequenced and published (Schneiker Set al., “Genome sequence of the ubiquitous hydrocarbon-degrading marinebacterium Alcanivorax borkumensis.”, Nat Biotechnol, 2006 August;24(8):997-1004) and is available from the NCBI Genome Project Database(NC_(—)008260; GenBank AM286690). A. borkumensis SK2 is a marinebacterium that uses oil hydrocarbon as its exclusive source of carbonand energy. It grows on predominantly alkanes and often becomes thedominant microbe that may comprise 80% of the microbial community in anoil contaminated environment. Bacteria of the Alcanivorax genus belongto a larger group of hydrocarbonoclastic bacteria that also includes thegenera of Cyclolasticus, Marinobacter, Neptunomonas, Oleiphilus,Oleisprira and Thalassolituus. These bacteria are able to metabolizeboth aliphatic and aromatic hydrocarbons. These bacteria represent agood source of genes that are involved in hydrocarbon utilizationpathways.

With the advent of rapid and inexpensive genome sequencing, thebacteria's genes and their roles in hydrocarbon degradation, surfactantproduction and gene regulation are becoming available. Databases such asGenBank, Swiss Prot, and others provide extensive genomic sequence datafrom these hydrocarbon degrading microbes. This data can be searchedwith computer programs such as BLASTX and BLASTN at the National Centerfor Biotechnology Information. In addition, the use of PCR amplificationbased on probes with complementary sequences from the highly conservedsequences for enzymes, known to be needed for hydrocarbon degradation,can be used to isolate and characterize homologues genes from newmicrobes from oil contaminated sites and oil reservoirs. This can bedone to analyze the change in protein sequence that has evolved to adaptto different environments. Such methods would be a useful way to findenzyme sequence modification that evolved as adaptation to the specificenvironmental. For example, microbes isolated from a high salt oilreservoir could contain enzymes that could degrade hydrocarbons in ahigh saline environment. These enzyme sequences could be compared tohomologues enzymes that function in a low salt environment to understandhow to modify the sequence of proteins to be soluble and functional inan obligate halophile.

In the case of A. borkumensis SK2, various gene clusters have beenidentified that are required for; the degradation of short chainalkanes; the degradation of large alkanes up to 32 carbons in length;and the degradation of branched aliphatic and alkylcycloalkanes. Part ofthis process of hydrocarbon metabolism is the production of surfactantsfor the emulsification of various types of hydrocarbons. In the case ofthe smaller or lower molecular weight chain hydrocarbons, theemulsification aids in the transfer of the hydrocarbon across the cellmembrane so that it can be metabolized. Therefore, this gene clusterincludes genes useful for mobilization of oil which could be transferredto a host microbe. It also contains genes that are not wanted, such asgenes that code for the proteins that are needed for the transfer ofsmall alkanes into the cell for further breakdown and consumption.According to the present invention, the unwanted genes are nottransferred to a host microbe or are inactivated or repressed. Genesrequired for the metabolism of larger alkanes, or alkylcycloalkanes, orpolycyclic aromatic hydrocarbons would be candidates for transfer intohost microbes.

The lack of enzymes that oxidize short chain alkanes would block or atleast slow down the transfer of small alkanes across the membrane. If anobligate halophile is engineered to serve as the basis for thisinvention it can be given the genes that are required for degradation ofall the petroleum components that would be beneficial to remove from theproduced oil. To compensate for the slower metabolism of these largerand more recalcitrant hydrocarbons, the host halophile may also need asoluble carbon and energy source to maintain growth.

Many of the well studied oil degrading microbes are considered to be auseful source of genes sequences required for proteins and pathways tomake useful products for the mobilization of petroleum oil. In oneexample, one or more bio-surfactants may be secreted by the cells to aidin the emulsification of the oil droplets so that the oil can beabsorbed through the cell wall. Several strains of Bacillus subtilis andBacillus licheniformis have been used to produce a lipopeptide namedsurfactin at commercial scale. This lipopeptide is also useful as anemulsifier and an antibiotic. Bacteria can be used to produce thesesurfactants in fermentation with manipulation of environmental andnutritional factors to increase yield as described by Cooper et al. in1981, Appl. Environ. Microbiol. 42:408-412, by Javaheri et al. in 1985,Appl. Environ. Microbiol. 50:698-700, and Guerra-Santos et al. in 1986Appl. Microbial. Biotech. 24:443-448. More recently, Mulligan et al.reported in U.S. Pat. No. 5,037,758 a genetically modified strain of B.subtilis ATCC # 21332 with a mutation at a site in a gene of wild typeB. subtilis that is able to produce surfactin at much higherconcentrations than the wild type. Therefore, by using gene transfertechniques these well studied genes, encoding for surfactant production,can be transferred into various host cells and the production ofsurfactant controlled.

There are many types of bio-surfactants that are useful in theemulsification of oil. Pseudomonas aeruginosa and other species canproduce rhamnolipids, which have a different structure than surfactin,but still function to immobilize oil. Other surfactants, such assophorolipid and mannosylerythritol lipid and glycolipids are producedby various strains of Candida. Over 200 different variations ofbio-surfactants have been reported. The different surfactant structureshave varying degrees of effectiveness depending on the pH, saltconcentration and other environmental factors. Because both the sequenceand amino acids will affect the solubility of the seven amino acidpeptide of the lipopeptide surfactin there are many millions of possiblecombinations. It is therefore likely that new strains of bacteria withunknown genes controlling the production of different bio-surfactantsthat are better at mobilizing oil for some reservoir environments couldbe isolated.

Surfactants and bio-polymers that are functional at high saltconcentrations and that are synthesized by either Archaea or bacteriathat are adapted to high salt cytoplasm are particularly preferred. Theisolation of microbes from environments extremely high in salinity areuseful because they may contain genes that code for surfactants andbio-polymers that would be useful in the selection and engineering ofhalophilic microbes for MEOR processes. Recently, Kebbouche-Gana et al.in J. Ind. Microbiol. Biotechnol (2009) 36:727-738 reported isolatingfive halophilic Archaea that are able to produce surfactants above 15%salts and that are able to metabolize both simple carbon sources andalso diesel. The five halophiles isolated by Kebbouche-Gana et al. areexamples of microbe that are likely to have genes containing sequenceinformation regarding enzymes that have adapted to high saltconcentrations. A technique for surfactant quantification and screeningfor biosurfactant-producing microorganisms is given by Bodour A A andMaier R M in J. Microbiol. Methods (1998) 32:273-280 and is incorporatedby reference. Halophilic Archaea such as Haloferax medierranei have beenreported to produce extracellular polysaccharides by Anton J. et al inAppl. Envirn. Microbiol. (1988) 54:2381-2386. In addition, halotolerantand thermotolerant bacteria have been reported to produce salt andthermo resistant bio-polymers in Appl. Envirn. Microbiol. (1986)51:1224-1229 by Pfiffner, S. M. et al. These isolated microbes arepossible sources of genes, that with some degree of modification couldbe transferred into a halophile that could be used as part of a culturedesigned for high salinity MEOR processes. In addition, similarisolation techniques could be used to isolate halophilic Archaea andbacteria from high salinity oil reservoirs that could provide bothmicrobes and genetic information that would be useful in selecting andengineering a culture of microbes for high salinity applications ofMEOR.

The process of identifying new genes based on DNA sequence similarity orhomology to known gene sequences of similar function is well known tothose skilled in the art of molecular biology. Several methods have beenused in the past. One method is to make probes of complementary RNAsequence with florescent or radio-labeled tags that will bind to mRNA ofthe genes being expressed by the bacteria in the environment. A secondtechnique is to use PCR amplification of DNA isolated from theenvironment with probes made from conserved sequence regions of thesought after genes. A third method used for screening for bioactivitiesis taught by J. M. Short in U.S. Pat. No. 6,030,779. With any of thesemethods, new gene sequences can be isolated from environments ofinterest such as an oil reservoir that is currently undergoing asuccessful MEOR operation. An alternative is an extreme environmentsimilar to one that might be encountered in an oil reservoir such ashigh salt concentration. For example, genes that code for abio-surfactant that is particularly well evolved for high temperaturemay be isolated from microbes in a high temperature oil reservoir. Thesegenes may then be transferred into a halophilic microbe that could use asoluble carbon source and express a surfactant to immobilize oil in areservoir that is both high in temperature and also high in salinity.

Another group of oil degrading microbes that are a good source of genescoding for useful products are microbes that can only metabolize highermolecular weight or complex hydrocarbons. For example, Banerjee et al.in U.S. Pat. No. 5,013,654 (1991) reported strains of an aerobicbacterium that will grow on paraffins of chain length longer than 12carbons. They also isolated a mutant strain of P. aeruginosa SB-1,designated SB-3, which has the property of growing on solid paraffins incrude oil of 20 carbons or more, but will not grow on the liquid lighterchain hydrocarbons. Bacteria such as SB-3, which was deposited in theAmerican Type Culture Collection, Washington D.C. as P. aeruginosaA.T.C.C. 39615 contain the genes for extracellular degradation andmetabolism of heavy petroleum oil. These genes, and others isolated bysimilar means, can be transferred into a host microorganism that is ableto thrive in the extreme environment of an oil well. This ability,combined with the ability to produce various surfactants andbiopolymers, and without the ability to consume light oil is useful inrecovery of light weight petroleum. If such microorganisms could alsouse a simple carbon source they could grow fast and predominate themicro-flora of a reservoir. In addition the engineered microbe could begiven a toxin resistance gene in addition to salt or temperaturetolerance as a further competitive advantage over the indigenousmicrobes that could consume the light weight oil.

More recently, Lei Wang et al. (PNAS Mar. 27, 2007, vol. 104(13):5602-7) reported the genomic sequence of a thermophilic Geobacillusisolated from a deep oil reservoir that could grow on long chain alkanesup to C36, but was unable to grow on short chain alkanes. Their analysisof the genomic sequence showed that it did not contain any homologousgene sequences to the AlkB genes that code for the membrane boundmonooxygenases that oxidize short chain alkanes. This group alsoreported a soluble and extracellular enzyme for the oxidation of longchain alkanes. This is another example of a source of genes that can beincorporated into a microbe for an additional source of energy andcarbon without the detriment of consumption of light weight oil.Microorganisms isolated from heavy oil reservoirs or other oilcontaminated locations are likely to contain genes for all types ofhydrocarbon metabolizing pathways. The membrane bound monooxygenasesevolved in the transport and oxidation of light chain alkanes can bedifferentiated from extracellular enzymes required for the oxidation ofhigher molecular weight hydrocarbons which are too large and insolubleto transport across the cell membrane.

In addition to degradation of high molecular weight paraffins, microbesmay be able to degrade other unwanted hydrocarbons in petroleum oil.Polycyclic aromatic sulfur containing hydrocarbons such as thiophenesand dibenzothiophenes (DBT) can be present in petroleum at high enoughlevels that they are toxic to bacteria and detrimental to the refiningprocess. The presence of sulfur compounds in oil will reduce the valueof the recovered oil. The sulfur is generally removed prior to refiningby expensive chemical processes. The need for a lower cost process hasencouraged the development of biological processes based on severalspecies of bacteria that have been isolated that can degrade thesesulfur compounds.

In one example, Rhodococcus sp. Strain IGTS8 converts DBT to2-hydroxybiphenyl (HBP) and inorganic sulfur. The pathway requires twomonooxygenases and a desulfinase. In addition to sequencecharacterization, these enzymes have been improved by site directedmutagenesis to broaden the substrate specificity to include thiophenesand benzothiophenes. A more detailed description of the pathway is givenby Gray, K. A. et al in Nature Biotechnology 14; 1705-1709 (1996).Although this biodesulfurization of crude oil is efficient at removingsulfur with little reduction in fuel value its wide spread use has beeninhibited by the cost of operating large stirred and aerated reactors.The reactor cost problem can be eliminated by transferring the genesthat code for the proteins in the metabolic pathway into a hosthalophile in such a way that they function in an oil reservoir todegrade the sulfur containing hydrocarbons at the same time as oil isbeing released from the reservoir during waterflood MEOR.

In summary, the preferred microbe of the present invention (i) containsfunctional genes for the metabolism of high molecular weight or the lessdesirable hydrocarbons; (ii) lacks functional genes for the transportand oxidation of short chain alkanes at the cell membrane; (iii)contains the genes for the production of useful compounds for oilrecovery and mobilization such as surfactants and polymers; and (iv) isregulated to express the useful compounds at high levels even if given asimple carbon nutrient supplement. In a preferred embodiment, themicrobe is capable of functioning and growing in the extreme high saltenvironment of a petroleum reservoir. In another preferred embodiment,the microorganism is capable of functioning in either an aerobic or alimited oxygen environment. With prevention of short chain alkanedegradation the interdiction of air containing oxygen is able to speedgrowth and oxidative degradation of large high molecular weighthydrocarbons into smaller light weight hydrocarbons for the reduction ofoil viscosity. In addition the petroleum's content of sulfur andnitrogen can be reduced, if desired.

(2) Selection of Extremophiles

Microorganisms that thrive in environments that would kill mostorganisms are referred to as extremophiles. These environments maycontain organisms from all three domains although generally are almostexclusively populated by prokaryotes, many which belong to the Archaeadomain of organisms. One type of extreme environment is a hypersalineenvironment. Naturally occurring aquatics of extremely high saltconcentration have existed on the earth many million years. Currentexamples are salt lakes or the Dead Sea as well as some petroleumreservoirs, all of which have existed for many years at saltconcentrations several times higher than sea water. This has allowed theevolution of organisms that have adapted to these consistently high saltconcentrations. The same is true for high temperature aquaticenvironments such as deep ocean thermal vents, hot springs and deeppetroleum reservoirs. Microbes have also been found in acid rockdrainage as low as pH 1 or alkaline soda lakes in Africa and other partsof the world as high as pH 11. Microbes have also developed genes codingfor the resistance to these toxic or extreme environments through anevolutionary process that may have taken many millions of years. Someresearchers believe that theses extreme environments are morecharacteristic of the earth when life first began.

These extreme environments can provide sources of both microbes andtheir genetic information that can be transferred into the appropriatemicrobes that are capable of functioning in the extreme environment ofan oil reservoir. In the case where a petroleum reservoir containsindigenous microbes that are detrimental to oil recovery (light oildegraders) the salt concentration could be adjusted to a salt level thatis toxic to the indigenous microbes but is still within the preferredrange of environmental conditions favorable to the engineered strain.This adjustment can be by waterflooding with a fluid as part of the oilrecovery process. Therefore, the selection of halophilic microbes foruse in oil recovery is the basis of this method. This invention providesmethods of developing a culture of microbes that will carry out an oilrecovery process without the unwanted consumption of short chainalkanes. In prior methods of MEOR, by simply stimulating the indigenousmicrobes in petroleum reservoirs the consumption of short chainhydrocarbons could cause reduction in oil viscosity.

High salt environments can be inhabited by halophilic microbes from bothdomains; bacteria and Archaea. Aquatic environments can be variable insalt concentration or consistently high in salt. Halotolerant (high saltconcentration tolerant) microbes can inhabit both variable andconsistently high salt aquatic environments. These are different thantrue halophilic microbes (salt-loving) that inhabit only consistentlyhigh salt environments. Currently there are about a 100 microbes thathave been isolated and studied from high salt environments.

Microbes that can live in a salt water environment have adapted to thehigh osmotic pressure by using two different mechanisms. In the firstprocess, the cell responds to an increase in salt or a drop in waterconcentration outside of the cell membrane by making small organicmolecules. These small organic compounds will reduce the waterconcentration inside the cell to equal the outside concentration. In thesecond mechanism, the high salt concentration (low water concentration)of the extra cellular environment is balanced by an equally high ionicsalt concentration (generally potassium chloride) within the cytoplasm.This is referred to as the “salt-in” adaptation. This salt-in adaptationrequires major changes to the structure of internal proteins and othercompounds in the cytoplasm of the cell. Microbes that have adapted tohigh salt by this mechanism are obligate halophiles and are unable tosurvive in a low salt environment.

The halotolerant bacteria make use of the small organic moleculemechanism to balance the osmotic pressure. Bacillus licheniformis JF-2(ATCC 39307) is an example of a microbe that is used to producesurfactant for MEOR and is halotolerant up to 8% NaCl (1.4 M NaCl).Their cytoplasmic water content is reduced by soluble organic moleculesto match the low water content of the external salt water environment.However, they use large amounts of energy to produce organic moleculesthat balance the osmotic pressure that is created by the high levels ofsalt in the environment. The halo-tolerant bacteria do not maintain highcytoplasmic salt levels because their intracellular proteins do notfunction in high salt. The use of organics to balance the osmoticpressure gives them an advantage in a changing salt concentrationenvironment. They can secrete small organic osmotic balancing moleculesquickly if the environmental salt concentration decreases. In addition,this type of salt tolerance has another advantage for the microbe. Itdoes not require changing a large number of the cell's proteins to adaptto changes in the salt concentration. For a microbe to become salttolerant it needs to acquire genes that code for the production of thesmall organic osmotic balancing molecules. These can be transferred bynatural horizontal gene transfer. However, this advantage comes with agreat energy cost needed for the production of the organic moleculesthat balance the external salt concentration.

(3) Obligate Halophiles

The first reported discovery of an obligate halophile was made byVolcani for his Ph.D. thesis in 1940. The halophilic Archaea wereisolated from the Dead Sea where they can exist at over 10 million cellsper ml. It was also discovered that these microbes could not grow inless than 1.5 M salt, which is about twice the salt concentration of seawater. After more than 30 year of research on these unusual microbes, anew domain of life known as the Archaea was proposed. Haloarchaea becamemodel organisms and were the first Archaea to be geneticallytransformed. Currently there are 7 haloarchaeal genomes listed on theUniversity of Maryland website (http://halo4.umbi.umd.edu/). In additionto haloarchaeal obligate haplophiles, there are bacteria that haveevolved the same mechanism of maintaining a high ionic cytoplasm.Currently 4 obligate halophilic bacterial genomes have been sequenced.Analysis of the proteomes of both the bacterial and haloarchaealhomologous enzymes have shown evidence of convergent evolution and LGTwhich has led to similarities in amino acid residues common to obligatehalophiles. It is therefore possible to make predictions ofmodifications or mutations to non-halophilic enzymes that would renderthem more soluble and functional in an obligate halophile cytoplasm.

Normally the proteins and other molecules that make up microbial cellswill not function at a high salt concentration. In order for enzymes andother compounds within the cytoplasm of a salt-in halophile to functionin the high ionic solution there must be changes made to the surfacecharges. Proteins can be altered in their number of basic and acidicamino acids that will make them more stable to high ionic solutions. Forthese microbes to have had to adapt to this different cytoplasmic saltconcentration they must have undergone significant changes to the aminoacid composition of their proteins and consequently the gene sequencesthat code for them. Analysis of the genomic sequences of these obligatehalophiles from both archaeal and bacterial examples indicate anincrease in the number of acidic amino acid residues and in particularaspartic acid and a decrease in basic amino acid residues (in particularlysine). In addition to an increase in the number of acidic amino acidresidues on the surface of the halophilic enzymes, there is generally adecrease in the number of hydrophobic amino acid residues, which leadsto a more flexible protein.

Changing one base pair of a three base pair codon is a single mutationand will not result in a charge change of the amino acid it codes for.Changing from a lysine to an aspartic acid requires a change in twobases in the codon (Lys, AAA or AAG to Asp, GAU or GAC). To make achange this radical, from a basic to acidic amino acid, requires adouble or triple mutation of the codon's base pairs. These radicalchanges are the type found in homologous proteins as seen in thecomparison of halophile to non halophiles or halotolerant microbes. Theyare unlikely to occur from simple point mutations, which would notresult in such large charge differences. Therefore, this type ofadaptation would be extremely slow and not likely to occur in a speciesfor many years.

Adaptation is unlikely to occur as a result of simple horizontal genetransfer from a non-halophilic microbe into an obligate halophile. Genesfrom non-halophiles or from low salt cytoplasm halotolerant microbesmust first be modified so that the proteins they code for will be stableto high salt concentrations. This key feature of the salt-in or obligatehalophiles can be the basis for a means that prevents the unwanted genetransfer from most other bacteria. If the indigenous is moderate in saltconcentration, the genes from other indigenous microbes in the petroleumreservoir will not function in an obligate halophile. If an undergroundoil reservoir contained a large population of microbes that couldmetabolize the light weight oil these unwanted genes could not be pickedup by the engineered obligate halophilic microbe. To be functional theindigenous genes would have to go though major changes so that theywould be functional in the high ionic cytoplasm of the salt-inhalophile. Generally, oil reservoirs that are either low in saltconcentration or are subject to variation in salt concentration would beunlikely to contain microbes that could contribute functional genes toobligate halophiles.

In the special case, where an oil reservoir was high in salinity andcontained indigenous halophilic microbes of the salt-in mechanism typeand that also had genes that coded for light chain alkane degradationenzymes, LGT could potentially be a problem. In this case, thesemicrobes should be killed off and eliminated from the reservoir beforeintroducing the high salt culture selected or engineered for oilrecovery. One method of eliminating indigenous halophiles is by freshwater-flooding of the reservoir. Generally obligate halophiles will notsurvive an exposure to water as low in salt as sea water. In addition,various toxic chemicals or biocides could be added to the low saltwater-flood to help eliminate indigenous microbes that could function athigh salt and that could consume short chain hydrocarbons. After thereservoir is cleared of indigenous high salt microbes, the engineeredshort chain alkane deficient culture can be added with controlled saltconcentration water-flood fluid.

In certain embodiments of the present invention, new genes are added toa host halophilic microbe. After a microbe is selected for use in a highsalinity reservoir it may be desirable to add genes for the degradationand use of high molecular weight hydrocarbons and/or the production ofsurfactants and polymers. If these genes are transferred from othernon-obligate halophiles it may be necessary to modify the genes for highexpression and function of the encoded enzymes in a high saltenvironment. This can be done by a combination of rational proteinsequence design and site directed mutagenesis. Therefore, the proteinsand enzymes required for the production of a surfactant or a hydrocarboncleaving enzyme useful for oil emulsification, can be engineered into atrue halophile after the gene sequences are changed to make the proteinsmore functional at high ionic salt concentrations.

Madern, D. et al Biochemistry (2000) 39: 1001-1010 reported on thestructural differences of both malate and lactate dehydrogenases foundin haloarchaea. Later, Ebel, C. et al in Biochemistry (2002) 41:13234-13244, reported on solvent interactions of halophilic malatedehydrogenase. A recent comparison of amino acid use differences betweenobligate halophiles is provided by Sandip Paul et al. in Genome Biology2008 9:R70. From analysis of these model enzymes found in both obligatehalophiles, mutations can be made to genes isolated from non-halophilesso that those genes will be expressed in halophiles at high yield andwill be functional at high salt. Also a number of these mutations can beevaluated for expression and enzymatic functionality in a halophile suchas Halobacterium sp. NRC1 or Haloferax volcanii using molecular genetictools such as the “pop-in-pop-out” method reported by Bitan-Banin etal., 2003 J. Bacteriol 185: 772-778 or Wang, G. et al., (2004) J.Bacteriol 186: 3187-3194.

In accordance with the present invention, if needed, the proteins andenzymes required for the production of a surfactant or a hydrocarboncleaving enzyme useful for oil emulsification are engineered into a truehalophile after the gene sequences are changed to make the proteins morefunctional at high ionic salt concentrations. The mutated sequences areevaluated for expression and activity in a high salinity environment. Inaddition, the engineered halophilic microbes will not acquire the lightoil consumption genes from the indigenous microbes existing in thereservoir, because the proteins encoded for are functional in the lowionic cytoplasm of a halotolerant microbe and are unlikely to functionin the high salt cytoplasm of the engineered “salt-in” halophile.

The “salt-in” obligate halophiles cannot tolerate a drop in saltconcentration, unlike the halotolerant bacteria which can quickly adaptby secreting small organic molecules, like glycerin as the salt levelchanges. In many cases, even a drop down to the concentration of seawater is enough to destroy the cell wall and kill the “salt-in”microbes. Even a slow adjustment over years would kill off the truehalophiles because of the huge changes in gene sequences that would berequired. Therefore, halophilic salt-in microbes have an advantage inthat they can thrive in a high salt environment without undue energyrequirement for maintaining osmotic pressure, if the salt concentrationdoes not drop below the toxic limit for the halophiles.

Therefore, a halophilic Archaeon is a good host microbe to engineer foroil reservoirs that can be maintained at high salt concentration at alltimes. It has a competitive advantage because it does not need toproduce large amounts of small organic molecules to balance the osmoticpressure. Also, the engineered microbes can be readily killed byreducing the salt concentration of any process solution before leavingthe site. As an added benefit, this has a regulatory advantage inmeeting EPA requirements under Toxic Substances Control Act (TSCA) onboth release and survival in the open environment. The details formethod of containment and requirements for the proper disposal arepublished in the Federal Register of Apr. 11, 1997.

Conversely, halotolerant microbes are preferred if the process wereexpected to encounter changes in the salt concentration. Thehalotolerant engineered microbes will be able to tolerate changes andfluctuations in salt levels which can be used to kill off truehalophiles that might interfere with the engineered microbes. In thatcase, the halotolerant culture needs to be maintained on a preferredcarbon source to provide enough energy and carbon for salt resistance.

Examples of Halophilic Microbes

The first genome of a “salt-in” halophilic Archaea, Halobacterium spNRC-1 (NC_(—)002607, NC_(—)002608 and NC_(—)001869), was completelysequenced in 2000 (L. Hood et al PNAS 97 pp 12176-12181). It became amodel microorganism to study. Although it is an aerobic mesophilicArchaeon, its ease of culturing and the ability to manipulate andreplace genes make it a good host or gene recipient microbe to develop alaboratory model for expression of surfactant and bio-polymers useful inoil recovery at high salt. It has a 2,571,010 by genome that codes for2,630 predicted proteins, many of which have known or predictedfunctions. Since then, the genomes of at least five more of the“salt-in” halophiles have been sequenced. Their genomes and proteomescompared to some other non halophiles by Sandip, Paul, et al. in GenomeBiology 2008 found online at (http;//genomebiology.com/2008/9/4/R70).These well studied extreme salt loving organisms could serve as a hostmicrobe to engineer for the production and secretion of usefulmetabolites that would emulsify and aid in the recovery of oil frompetroleum reservoirs.

Halophiles may be isolated from oil reservoirs. A typical oil reservoircan be not only high in salt, but low in oxygen and at elevatedtemperatures. The host halophile should be chosen to use an alternateelectron acceptor such as nitrate and be able to grow at 40 to 80degrees Celsius. These microbes would be found in high salt environmentsthat were also deep or low in oxygen and at high temperatures. Deep oilreservoirs that are high in salt is a likely environment to isolateuseful microbes from that could be engineered into producing useful oilrecovery metabolites without consuming oil. Needed genes such as theability to grow on a singles carbon source could be added if the microbelacked them.

Halobacterium sp. R-1 (NC_(—)002607, NC_(—)002608 and NC_(—)001869), isan extreme halophile similar to Halobacterium sp. NRC-1. Its 2.8 Mbpgenome was completed in 2008.

Haloferax volcanii (NC_(—)013967), is a moderate halophile isolated fromDead Sea mud. It grows optimally, with a generation time of about 4hours in rich medium containing 1.5-2.5 M NaCl at 45 degrees C. Itrequires at least 0.02 M Mg and is tolerant of up to 1.5 M Mg. It cangrow more slowly in minimal medium with glucose as a single carbonsource. It genome was completely sequenced in 2006. Halofexax volcaniiis widely used for genetic experimentation as is Halobacterium sp. Amore detailed description of this microbe is given by Berquist, Mullerand DasSarma in Method in Microbiology Vol. 35: 649-679.

Haloarcula marismortui (ATCC 43049), is an Archaeon metabolicallyversatile and an extreme halophile from the Dead Sea. It has a 4.3 Mbpgenome that was completely sequenced in 2004.

Haloarcula quadrata, (in culture collection as AB010964, AB010965,DSM11927) growth in 2.7 to 4.3 M NaCl, pH 6.5 to 7.0, optimumtemperature 53 deg. C., anaerobic growth on nitrate, growth on a singlecarbon source. This culture was isolated in Sinai, Egypt. Ref. Oren etal. 1999, Int. J. Syst Bacteriol 49 1149-1155

Halogeometricum borinquense (in culture collection as AF002984 and ATCC700274) growth in 1.4 to 5.2 M NaCl optimum range 3.4 to 4.3 M NaCl, pH7 optimum temperature 40 deg. C. anaerobic growth on nitrate, growth ona single carbon source. Isolated from a saltern in Puerto Rico. Ref.Montalvo Rodrquez et al. 1998 Int. J. Syst. Bacteriol. 48: 1305-1312.

Haloferax denitrificans (in culture collection as ATCC 35960 and DSM4425) growth in 1.5 to 4.5 M NaCl optimum range 2 to 3 M NaCl, pH 6.7optimum temperature 50 deg. C. anaerobic growth on nitrate, growth on asingle carbon source. Isolated from a saltern in California USA. Ref.Tomlinson et al 1986 Int. J. Syst. Bacteriol. 36:66-70

Haloquadratum walsbyi, is a square shaped extreme halophile isolatedfrom solar salterns with a 3.2 Mbp genome that was sequenced in 2006.

Alkaliphilic Archaeal Halophiles:

Alkaliphilic halophiles can be found in hypersaline soda lakes such asLake Magadi in Kenya, Wadi Natrum lakes in Egypt and soda lakes inChina. These could be engineered to produce bio-surfactants and otherbiological oil recovery compounds that were effective at alkaline pH.Generally alkaline pH is better for oil emulsification. Increasing thepH of the flood water can be done by adding caustic soda and would havethe added advantage of suppressing the growth of endogenous microbesthat might interfere or have detrimental effects on the quality of theoil produced.

Halothermothrix orenii is an anaerobe isolated from a Tunisian salt lakethat grows in 3.4 M NaCl (20% salt) at 68 deg. C. Ref. Cayol J-L et al1994 Int. J. Syst. Bacteriol. 44: 534-540.

Natronobacterium magadii and N. gregoryi are alkaliphilic halophilesbut, not thermophiles that have been isolated from Lake Magadi in Kenya(ref. Tindall et al 1984 ATCC 43099 and 43098). They have a pH optimumof 9.5 and a salt range of 2.0-5.2 M NaCl.

Natronomonas pharaonis (NC_(—)007426, NC_(—)007427, and NC_(—)007428),is an alkaliphilic extreme halophile isolated from a soda lake. ThisArchaea's 2.6 Mbp genome was completely sequenced in 2005.

Bacterial Halophiles:

Salinibacter ruber (NC_(—)014028, NC_(—)014030, NC_(—)014032), is anextreme halophile with a 3.6 Mbp genome that was sequenced in 2006.

Chromohalobacter salexigens (NC_(—)007963), is a moderate halophile thatsurvives on a variety of salts. Its 3.7 Mbp genome was sequenced in2006.

Halothermothrix orenii (NC_(—)011899), is a thermophilic bacterialhalophile with a 2.7 Mbp genome sequenced in 2006. This bacterium wasisolated from sediment of a Tunisian salt lake and grows optimally from50 to 100 g per liter NaCl at 60 degrees C.

Halorhodospira halophile, is a bacterial extreme halophile that canoxidize sulfur. Its 2.7 Mbp genome was sequenced in 2007.

In addition to the microorganisms listed above, a larger list isprovided by Enache, M. et al. in the International Journal of Systematicand Evolutionary Microbiology (2007), 57:2289-229, which is expresslyincorporated by reference herein. In addition to the halophile listedabove other halophiles can be selected from culture collections orisolated from high salt environments.

Further details of the invention are provided in the followingnon-limiting examples.

All references cited throughout this disclosure and the references citedtherein are expressly incorporated by reference herein.

EXAMPLES Example 1 Step 1: Microbe Isolation, Characterization andImprovements Site Selection

Environments of consistently high salt concentrations (salinitiesexceeding 100,000 ppm total dissolved solids) are best for isolation ofobligate halophiles. Sites also containing liquid hydrocarbons such aspetroleum oil fields or waste oil/brine disposal pits are goodcandidates for microbes that are both halophiles and also have theability to metabolize various types of hydrocarbons. Microbes selectedfor use in MEOR should be able to function in a low oxygen environment.Facultative anaerobes are ideal host microorganisms. Especially good aremicrobes that can use nitrate as an election acceptor. Aerobic microbesmay be used in applications where large amounts of air can be injectedwith the waterflood fluid. Microbes isolated from these environments canalso be a source of genes and gene sequence information that can be usedto genetically modify a culture of microbes, which can be tested andused on oil reservoirs of high salinity or where brine is used aswaterflood.

In one example, following the method described by S. Kebbouche-Gana, etal., reported in the J. Ind. Microbiol. Biotechnol. 2009 36:727-738,isolating five strains of Halobacteria or Haloarchaea that were able toproduce bio-surfactants, samples are collected at 1-m intervals from ahigh salinity pond. The microbial isolates are cultured in a standardmedium containing 125 g of NaCl, 160 g of MgCl₂ 6H₂O, 5.0 g of K₂SO₄,0.1 g of CaCl₂ 2H₂O, 1.0 g of yeast extract, 1.0 g of casamino acids and2.0 g of soluble starch. The pH is adjusted to 7.0 with NaOH. Thismedium is also increased to 3.5M NaCl (203 g/l) and 5% v/v diesel oil.Cultures are grown at 40° C. with shaking at 200 strokes per minute.Various carbon sources such as glucose, fructose, arabinose and sucroseare tested by omitting starch and reducing yeast extract and casaminoacids to 0.25 g/l. Growth is monitored by optical density at 600 nm. ThepH of each culture is determined and a drop in pH to below 6.0 isconsidered evidence of acid production.

Surfactant production is monitored by a drop in surface tension.Screening of colonies for surfactant production is performed by thequalitative drop-collapse test described by Jain et al. (1991) J.Microbial Methods 13:273-280. Two microliters of oil are applied to wellregions delimited on the covers of 96-well microplate (Biolog, Hayward,Calif.) and left to equilibrate for 24 hours. The oil used for the testcan be motor or petroleum oil characteristic of the intended reservoirfor MEOR. A liquid sample is removed from each of the isolated straincultures after 7 days of incubation. After centrifuging for 5 minutes at12,000 g to remove cells, 5 microliters are added to the oil coated wellregions. The drop size is observed after 1 minute with the aid of amagnifying glass and compared to a sterilized negative control. A dropdiameter at least 1 mm larger than the control is considered anindication of surfactant production. The positive isolates are furtherevaluated for oil emulsion-forming ability and emulsion stability.

Surface tension reduction depends on the chemical and physicalconditions such as temperature, salinity and pH. For laboratory testingthese conditions should be tested over the range that is expected for anoil reservoir. For evaluation of chemical surfactants, a simple labscale test was developed by Larry W. Lake, (from a course, “Fundamentalsof Enhanced Oil Recovery”, at the University of Texas). This test can bemodified to evaluate the effectiveness of surfactant for EOR. For themodified test, a number of tubes are filled with equal volumes of oil(ideally the petroleum oil that is typical of the reservoir to betreated with MEOR) and an aqueous phase that represents the fluid to beused as the waterflood buffer. The salinity, temperature and pH can bevaried over a range that would be economical at a commercial scale for awaterflood fluid used as part of the process. To the aqueous fluid,various size aliquots of cell suspensions are added to each tubeexperiment. It is best to use cells that are in exponential growthphase, or are at a phase when surfactant production is high. The tubesare vigorously shaken and left to form emulsion layers. After severalhours the middle layer between the top oil layer and the bottom aqueouslayer is measured. The larger the middle layer, the better the aqueousfluid, which contains microbes and the surfactants they make, is atreducing the interfacial tension (IFT) and the better it will be atimproving oil production. These results can be tabulated to identify thebest microorganism strain in terms of size of middle layer per unit ofcells or volume of cell broth. The selection of the best strain willdepend on the physical and chemical condition of the fluid used as awaterflood buffer. Some strains may be best for high salinity fluids andothers may be best at lower salinity fluids. The selection of chemicaland physical conditions will depend on economic and environmentallimitations of the petroleum reservoir.

Bio-consumption or utilization of the hydrocarbon liquid added to themedia is often linked to the production of surfactant. That is becausemicrobes that metabolize oil will often make surfactants to aid in theuptake of oil. Microbes that produce surfactant are also likely toexpress high levels of enzymes that are needed for the degradation ofliquid petroleum. Ideally one would like to find a microbe that couldproduce surfactant without also consuming oil, especially the shortchain alkanes. This is difficult because the genes for both oilconsumption and surfactant production are generally clustered togetherand controlled by the same promoter. However, the genetic informationcontained in microbes, that have the capability of doing both, isuseful. Knowing the sequence of the genes that code for monooxygenases,which are generally the enzymes that start the alkane metabolism, isuseful information for the modification or elimination of the shortchain alkane metabolizing capabilities.

Step 2: Isolation of DNA and Genes Needed for Surfactant Production andLiquid Oil Consumption

Microbial strains selected for high and effective surfactant productioncan be further characterized by gene sequencing. DNA is extracted frompoly-carbonate filters as described by Minz et al. (1999) Appl. Environ.Microbiol. 65: 4666-4671. This procedure was modified by Kebbouche-Ganaet al. The DNA was electrophoresed, excised from the gel and purifiedwith a jet sorb gel extraction kit (Genomic DNA purification systemPROM, EGA). Purified DNA from selected strains are amplified withspecific 16 s rRNA archaeal primers (5′-TTCCGGTTGATCCYGCCGGA-3′(SEQ IDNO: 1) and 5′ YCCGGCGTTGAMTCCAATT-3′ (SEQ ID NO: 2)). 16 s rRNA sequenceinformation can be aligned with rRNA sequence from known Halophiles forgenera and family identification.

DNA or mRNA probes can be based on known genes from an organism thatproduces a surfactant. One example is Pseudomonas aeruginosa, whichproduces rhamnolipid. The synthesis of this glycolipid is by sequentialglycosyl transfer reactions. The genes involved in rhamnolipidbiosynthesis are encoded on a plasmid, and their expression is regulatedby a quorum sensing system. A more complete review is given in Lang andWullbrandt (1999) Appl. Microbiol. Biotechnol. 51:22-32. Other species,such as Bacillus subtilis, produce surfactin, a lipopeptide whichcontains about 7 amino acid residues. Other microorganisms secretehigher molecular weight biosurfactants consisting of polysaccharides,lipoproteins, and lipopolysaccharides. Isolation and identification ofthe surfactants secreted by the isolated strains can be done by HPLCwith a mass-spec detection system. Identification of the chemical natureof the surfactants produced by each isolated strain can be usefulinformation for finding genes that are required for the surfactantproduction and secretion. For example, a glycolipid similar torhamnolipid would likely be dependent on genes similar to those involvedin its biosynthesis in P. aeruginosa. These genes from wellcharacterized microbes can be used to construct probes for findingsimilar genes in the halophilic isolates.

However, even if the surfactants produced by the halophilic microbes arecompletely new and unlike any other well studied surface activecompounds, other methods of gene isolation can be used. For example,correlating higher levels of mRNA with production of high levels ofsurfactant can be used to find needed genes. If the presence of alkanesinduces the production of surfactant, than the level of mRNA needed forsurfactant production will be increased. The use of DNA microarrays canidentify the increase in gene transcription into mRNA when surfactantproduction is induced. Sequencing of the cDNA made from the increasedmRNA can be used to identify the required genes sequences.

Based on this method, the identification of genes required for theproduction of surfactant production and the degradation of liquid oilcan be done by mRNA differential display. This method was used toidentify Cyclohexonone metabolism related genes (Brzostowicz et al.(2000) J. Bacterial. 182: 4241-4248). These mRNA techniques make itpossible to access regulated genes directly without purification of geneproducts. These approaches are based on comparisons of two cultures andthe identification of genes whose mRNA is more abundant when a metabolicpathway is induced. In the above example, if surfactant production isinduced by the presence of oil, then mRNA that codes for surfactantproduction as well as enzymes for oil metabolism will be at higherlevels compared to the uninduced culture. These techniques rely on thehybridization of DNA on membranes as described by Chuang and Blattner1993 J. bacterial. 175: 5242-5252. It was by this method thatBrzostowiez et al. that led to the discovery of the genes for twomonooxygenase enzymes responsible for the oxidation of cyclohexanone.This same technique can be used for the identification of genes codingfor proteins and gene products of halophilic archaea that lack enoughsequence homology to bind to probes constructed based on proteinsequence of non-halophilic or mesophilic homologous enzymes.

Probes may also be based on protein sequence of homologous enzymes withhighly conserved catalytic site and binding sites. In this case a shortdegenerate DNA probe is constructed to bind with any DNA that has thesequence of base pairs that code for the highly conserved amino acidresidue sequence.

Although all these method can be successful at isolating new genesrequired for surfactant production and liquid hydrocarbon oildegradation in halophiles, as more gene sequences are obtained fromobligate halophiles the faster the isolation of new genes isaccomplished.

Step 3: The Prevention and Modification of Short Chain Alkane Metabolism

The expression of genes required for the production and secretion ofsurfactants and the degradation of high molecular weight hydrocarbonsare beneficial to the mobilization of oil. It is the degradation ofshort chain alkanes and other low viscosity petroleum components that isvery detrimental to oil recovery. Therefore, if the genes of a microbecould be modified so that they do not metabolize light oil, theviscosity would decrease and recovery of petroleum would increase.However, this must be done in such a way that production of surfactant,which may be under the control of a single gene promoter, is not alsoprevented. With the loss of liquid oil metabolism, another utilizablecarbon source is needed to offset the loss of energy from the lightchain hydrocarbon metabolism. Often the genes needed for both liquidhydrocarbon consumption and surfactant production are clusteredtogether. Therefore, deactivating or removing the genes needed for shortchain alkane uptake must be done in such a way that the genes needed forhigh production of surfactant are not deactivated or down regulated.

One method of achieving this specific gene modification is homologuesgene replacement. A new gene replaces a similar wild type gene with amodified nucleotide sequence that codes for a protein with a differentamino acid sequence. This process can be used to make small changes toenzymes to change the catalytic efficiency or specificity of the enzyme.A change of one or two amino acid residues can make the new enzyme nolonger able to bind the same substrate or catalyze key steps in theconversion of substrate to product at the same rate. This process hasbeen used in many genetic systems wherein similar genes are replaced bymutated and by homologous recombination (Molecular Biotechnology editedby Glick and Pasternak, 2003, Chapter 8). Along with the mutated gene, aselectable maker is also incorporated so that new microbes that havetaken up the mutated gene can be selected. This process requires acertain level of genetic manipulation tools. Fortunately, a geneknockout system has been developed for the halophilic Archaea, Haloferaxvolcanii and Halobacterium salinarum based on the pyrE gene reported byBitin-Banin et al. in J. Bacteriol. 2003, 185: 772-778. This system hasbeen further developed and now four different selection principles areavailable (Allers et al. Appl. Environ. Microbiol. 2004, 70: 943-953)for Hf. valcanii. By using this technique or similar gene replacementtechniques with selectable makers, the monooxygenase genes isolated fromthe wild type halophiles can be replaced with modified genes sequences.

By this process or other genetic manipulation processes a number ofchanges can be made in the amino acid sequence of enzymes thatfacilitate the uptake or metabolism of light chain alkanes. This processcan be done by random changes to any amino acid in the enzymes sequence,by trial and error. The resultant enzymes with the amino acid changescan be tested for changes in substrate binding, substrate specificityand conversion to product rate. In general, most of the random changeswill have little effect, or will decrease the catalytic rate. Thisprocess is much easier if the three dimensional structure of the enzymeis known or can be determined by X-ray crystallographic analysis. Inthis example the structure of some alkane specific monooxygenases havebeen determined and are useful in predicting key amino acids to change.For example, by making point mutations of the amino acid residues at thebinding sites also known as histidine boxes, it would likely prevent orcause a reduction in the rate of alkane metabolism.

Changing any of the amino acid residues, especially the histidines, willaffect the ability of these enzymes to metabolize liquid hydrocarbons. Anumber of these modified enzymes can be evaluated in a model halophilichost such as Haloferax volcanii to determine the enzymes ability tofunction at high salt. Modified wild type halophiles with the mutatedalkane conversion enzymes can be evaluated at the laboratory scale fortheir ability to produce surfactant, but with limited ability to grow onoctane or diesel as a carbon source. From the group of engineeredmicrobes, the strains that achieve high levels of growth with aninexpensive carbon source, and that produce high levels of surfactant,and that consume the smallest amount of light molecular weight oil(C6-C8) are selected. The consumption of short chain alkane can bedetermined by analysis of remaining oil in the reaction vessel. A moresensitive method is with a carbon 14 isotope labeled alkane. Smallamounts of uptake of the isotopic carbon can be measured in the cells.Alternatively, the rate can be determined from the isotopic carbondioxide produced.

From this group of engineered microbes the selected halophiles aretested for their ability to mobilize oil in a laboratory scalewaterflood core sample test. This test consists of saturating areservoir rock core sample or a packed sand column with petroleum oil. Aflow of water or brine is then pumped through the core sample until thefree oil is washed out. Then the microbe culture in a growth buffer isintroduced into the core sample that still contains the residual oil.The core sample inoculated with microbes is left to incubate for one totwo weeks. After incubation, a flow of waterflood buffer is passedthrough the core and the amount of removed oil by this flow is measuredas a function of buffer flow volume. With this small scale laboratory,test the effectiveness of each of the engineered and wild type culturescan be measured and compared. The improved cultures should show anincrease in the rate and total amount of oil removed from the core.There should also be an increase in the number of microbes, indicatinggrowth in the high salt environment. However, this short test does notindicate oil or light chain alkane consumption because the time that themicrobes are in contact with the petroleum is too short and there is noeasy way to measure the total remaining oil.

Therefore, another approach is needed to determine the short chainalkane consumption. The conditions of the digestion should match thewaterflood drive buffer or fluid. It should contain the soluble carbonsource such as molasses that will be used to supplement growth. However,the soluble carbon source should not be a catabolite that will causerepression of alkane degradation pathway genes. A report of carbonsources that can cause repression of alkane degradation pathways inPseudomonas putida is given by F. Rojo et al. in the J. Bacteriology2003 185: 4772-4778. The incubation should be long enough (severalweeks) to measure degradation and loss of alkanes or a change in totalalkane hydrocarbon composition or a change to the relative amount ofvarious hydrocarbons if a mixture or sample of petroleum oil is used.The measure of an engineered or selected microbe that will be a goodcommercial candidate is that there is no decrease, or relative decrease,in the lighter weight hydrocarbon. As a comparison, this same test isperformed with the indigenous microbes isolated from the location or oilreservoir. A test using only the stimulation of indigenous microbesmight produce less oil, or produce oil with a larger high molecularweight fraction. The best cultures will be the ones that can produce themost surfactant and the most oil without decreasing the percentage oflight weight oil in the petroleum samples.

Example 2

In this example essentially the same procedure described in Example 1 isused to isolate halophiles that are able to grow in brine solution in anenvironment similar to an underground petroleum reservoir to isolate astrain that is able to utilize petroleum compounds of various types. Ifthe strain of halophile isolated is capable of metabolizing both shortand long chain alkanes, the gene for metabolizing short chainhydrocarbons is knocked out while maintaining the genes for longer chainalkanes. The ideal location for isolation of such halophiles is apetroleum reservoir with a brine solution of over 100,000 ppm of totaldissolved solids. In addition the temperature should not be more than80° C. so that the brine taken from the reservoir is likely to containmicroorganisms.

There are a number of species of microbes that have been reported thatonly degrade high molecular weight oil. For example, Banerjee et al. inU.S. Pat. No. 5,013,654, isolated a strain of Pseudomonas aeruginosaSB-1 and a mutant strain SB-3 that could only grow on paraffins(alkanes) of 20 carbons or more. Feng et al. in PNAS Mar. 27, 2007 p5602-5607 reported a non-halophile (Geobacillus thermodenitrficansNG80-2) that metabolized only alkanes over C15. This thermophile wasisolated from a hot petroleum reservoir in China. However, there arevery few reports of hydrocarbon degrading microbes that are alsoobligate halophiles.

Obligate halophiles that have the ability to metabolize higher molecularweight petroleum components are useful microorganisms to use for oilrecovery in high salinity brine oil reservoirs or where high salinitysolution is used as a waterflood fluid. In addition to being a good hostmicrobe to engineer, these microorganisms also provide a useful sourceof gene sequence information for the modification of enzymes fromnon-obligate halophiles to remain soluble and function in the high saltconcentration of an obligate halophile. Also, halotolerant microbes,that maintain osmotic balance with the production of small organicmolecules, often maintain slightly higher cytoplasmic saltconcentrations. Comparison of homologous protein sequences has shownthat these halotolerant microbes have evolved minor changes to theirproteins to accommodate the higher salt. The analysis of enzymes thatcatalyze the first step in the degradation of alkanes by halotolerantmicrobes such as a monooxygenase could provide insight into selectingamino acids for site directed mutagenesis to osmotically-adapt amonooxygenase to function in an obligate halophile. This sequenceinformation, in combination with the three-dimensional structureinformation, can be used to engineer a mesophilic enzyme to function athigh salt concentrations.

In the case where no high molecular weight alkane degrading halophilicmicrobes can be isolated, it is possible to modify and transfer a genefrom a non-halophile or halotolerant microbe into a halophile. Forexample a synthetic gene can be constructed to express a halophilicenzyme to degrade long chain alkanes based on a protein sequence from awild type non-halophile that can degrade long chain in lower saltenvironments. This can be done to replace a short chain monooxygenasegene like the one described in Example 1.

In a specific example, the wild type gene that codes for the long-chainalkane hydroxylase LadA isolated from G. thermodenitrificans NG80-2 (SEQID NO: 2 encoding polypeptide of SEQ ID NO: 3) is mutated to be moresoluble in high salt and function at a salt concentration of over 1.5 MKCl, which is typical of an obligate halophilic cytoplasm. In thisexample; the LadA protein sequence, the binding sites and the crystalstructure are reported by L. Li et al. in J. Mol. Biol. 2008, 376:453-465. In this specific example, the LadA protein sequence wascompared to other protein sequences in the protein data bank and foundto be a flavoprotein monooxygenase that utilizes dioxygen to insert anoxygen atom into the substrate. Based on X-ray data and molecularreplacement methods computer programs, Li and co-workers were able toreport the crystal structure with enough resolution to produce a modelof the LadA protein, the FMN coenzyme and the long chain alkanesubstrate complex. The binding cavity for the alkane is packed withhydrophobic amino acid residues which hold the 16-carbon or longerchain. The four polar residues, His17, Tyr63, Gln79, and His311 arelocated above the terminal carbon of the alkane to form the reactivesite. Directed mutagenesis was done to show that these four amino acidresidues plus Cys14 believed to be involved in a disulfide bridgeholding the dimer together are all required residues for activity. Tomake the mutants, nucleotides were changed in the LadA gene to replaceCys14 with an Ala, His17 with a Phe, Tyr63 with a Phe, Gln79 with a Leuand His311 with a Phe in the protein. Each of the mutants was clonedinto pET-28a(+) (Novagen, USA) with a 6×His tag at the N terminus andexpressed in E. coli BL21 (DE3). Cells were grown at 37° C. in 1 l of LBmedium containing 50 micro grams per ml kanamycin. Cells were grown toan OD₆₀₀ of 0.6-0.8, and were then continuously induced with 0.2 mMisopropyl-β-D-thiogalactopyranoside at 45° C. for another 4 h.

Protein isolation of each of the mutated enzymes that contained a singleamino acid residue change was done by cell lysis and disruption andsonication followed by centrifugation at 15,000 g. The solublesupernatant was applied to a Ni²⁺ chelating affinity column (1.5 ml ofNi²⁺-NTA agarose) pre-equilibrated with 20 mM Tris-HCl (pH 8.0) and 10mM NaCl lysis buffer. The contaminating proteins were washed off with 10bed volumes of 20 mM Tris-HCl (pH8.0), 10 mM NaCl and 20 mM imidazole.The target protein was eluted with 20 mM Tris-HCl (pH 8.0), 10 mM NaCl,and 200 mM imidazole with about 15 ml of buffer. The protein was furtherpurified by Resource Q anion-exchange and superdex-200 chromatography.The isolated proteins were assayed for activity by mixing with the longchain alkane substrate by the method described by Feng et al. with amodification. The reaction contained 50 mM Tris-HCl (pH7.5), 1 mMhexadecane, 1 mM each of FMNH₂ and MgSO₄, Remaining alkane substrate wasmeasured by gas chromatography (GC). The wild type protein was used as acontrol. Li and co-workers reported that all the mutant LadA wereinactive indicating that each of the five amino acid residues is neededfor catalytic activity.

The same method of mutating one residue at a time followed by activitymeasurement, or a variant thereof, is used to adapt the long chainalkane hydroxylase to function in a high KCl environment. However, toadapt this enzyme to function at high salt concentrations each mutantshould be assayed for activity at varying concentration of salt. Forexample, the rate of hexadecane conversion to hexadecanol should beassayed at; 100 mM NaCl, 100 mM NaCl and 200 mM KCl, 100 mM NaCl and0.5M KCl, 100 mM NaCl and 1M KCl. Any mutation that reduces activity atlow salt concentrations with no apparent increase in activity at highersalt concentrations is unlikely to be beneficial to osmotic adaptation.Mutations that do not decrease the rate of conversion at low saltconcentration can be useful when combined with several other mutations,which add negative charges or reduce rigidity of the protein, toincrease solubility and function at high salt.

The selection of amino acid residues to mutate for evaluation forosmotic adaptation is based on some general rules. The number of acidicresidues should increase on the surface of the protein molecule.Halophilic proteins generally have a lower isoelectric point as a resultof more acidic residues on the surface than their non-halophilichomologous proteins. The most common change is an increase in the numberof acidic residues by replacement of Lys with Asp. Another way ofreducing the isoelectric point is the insertion of a small domain orpeptide that contains an excess of acidic residues. These changes andadditions are to be performed on the amino acid residues on outersurfaces of the molecule or to peptide chains connecting domains. Theaddition of negative charges should not be at the hydrophobic bindingpocket or at positions that are conserved as basic residues inhomologous enzymes. Some possible changes to LadA to decrease theisoelectric point are listed in Table 1.

TABLE 1 Change to New AA Amino Acid Sequence Position Residue, a testedResidue in Lad A of Residue in Lad A in mutant Lys 48 Asp, Glu Lys 50Asp, Gly Lys 170 Asp Lys 195 Asp Lys 204 Asp, Glu Lys 263 Glu Lys 266Glu Lys 267 Glu Lys 276 Asp Lys 287 Glu Lys 295 Ala Lys 301 Gly, Ala Lys322 Asp Lys 343 Glu, Asp Lys 357 Asp Lys 358 Glu Lys 415 Asp, Glu Lys419 Glu Arg 262 Glu Arg 264 Gly Arg 413 Ser Arg 423 Ala Arg 432 Ala Arg434 Val

Another adaptation is a reduction in the number of large hydrophobicresidues (Ile, Leu, Met, and Phe), which are replaced with lesshydrophobic residues (Val, Thr). There is also an overall reduction inthe number of disulfide bridges. All these changes should avoid keybinding sites, reactive sites, important secondary structuredeterminates and conserved sequences. The goal of reducing the number ofhydrophobic residues and the number of disulfide bonds is to make theprotein molecule more flexible to function better in the high KClconcentration of the halophilic cytoplasm. All these changes can betested by expression and analysis of the enzyme with substrate invarious levels of salt concentration. It is possible that some changeswill decrease the activity at low salt concentrations, but will keep itthe same or increase the activity at higher salt concentrations. In theexample of LadA, some changes that reduce the rigidity of the moleculeare listed in Table 2.

TABLE 2 AA Residue in Sequence Position Change to new Lad A of Residuein Lad A AA in Mutant Cys-Cys 168 & 214 Ser, Ser (bridge) Cys-Cys 243 &282 Ser, Ser (bridge) Leu 152 Val Leu 171 Val Met 277 Thr Phe 278 ThrIle 281 Thr Met 293 Thr Leu 296 Ala Trp 303 Val Leu 305 Val Ile 332 ValIle 337 Val Met 341 Val Leu 344 Val

In addition to changes in amino acids, halophiles also have changes innucleotide use and GC-content of DNA. Accordingly, it is best toconstruct a synthetic gene based the abundance of GA, AC, GT and CGdinucleotides for stability at high salt concentration. The combinationof several amino acid residue changes or the addition of an extra domaincontaining negatively charged amino acids can be coded for by asynthetic halophilic gene designed for expression in a model halophile.Some codons that are frequently found in obligate halophiles are listedin Table 3.

TABLE 3 Codon Nucleotides Amino Acid Residue CGA, CGG Arg GUC Val ACGThr CUC Leu UGU Cys

Two examples of model halophiles are Halobacterium species NRC-1 andHaloferax volcanii DS2. A detailed description of their genetic systemsand methods for transformation is given by B. R. Berquist, J. A. Mullerand S. DasSarma in Methods In Microbiology Volume 35 2006 Chapter 27.These model halophiles are well suited to express synthetic halophilicgenes or wild type genes isolated from brine environments and likely tobe halophiles. Procedures for expressing protein variants in Haloferaxvolcanii are described by Reuter and Maupin-Furlow in Appl. Environ.Microbiol. 2004, 70: 7530-7538 and is incorporated by reference. Bythese methods, mutations or added extra domain can be tested forexpression and function in a high salt environment. In the aboveexample, after a number of mutations have been evaluated and that arefound to increase salt solubility without loss of activity, they canthen be tested in various combinations for function in a halophilicexpression system. Proteins adapted to function at high saltconcentrations may not be soluble or active at lower saltconcentrations. Therefore, in addition to expressing mutated enzymes ina model halophile it may also be necessary to isolate the protein in ahigh salt solution. This will require some changes to the protocol usedby Li et al. to isolate mutations of LadA expressed in E. coli. Thesechanges are likely to require adjustments to the binding and elution ofthe target protein from the Ni affinity column and the anion exchangechromatography. Gel filtration chromatography needs to be done at highsalt so that the proteins do no aggregate or precipitate. Alternatively,enzyme activity can be tested without isolation of the enzyme by directassay of the cell media to yield units of activity as a function of celldensity. Quantifying of target protein can be done by gelelectrophoresis.

After several of mutated forms of LadA are determined to be active at1.5 M or higher KCl, the synthetic genes are transferred into the hosthalophile for evaluation as a microbe to aid in the recovery of oil. Inone example, the host microbe is a halophile isolated from a high salinepetroleum reservoir. The gene coding for the high molecular weightalkane degrading enzyme can be used to replace the homologous genescoding for the low molecular weight alkane degrader by homologous genereplacement with a selectable marker. The transformed cells are thenselected and the marker removed. The transformed microorganisms are thengrown under conditions of salt and temperature similar to the reservoirenvironment that will be used in for oil recovery. These microorganismscan then be evaluated in an oil saturated reservoir core sample testdescribed above. In this test the surfactants and other compoundsproduced by the bacteria will dislodge a certain amount of oil over thebuffer control. This test does not, however, reveal the benefit ofhaving only high molecular weight petroleum degradation capability. Toevaluate that, another test is needed.

To test the usefulness of the new microbe a mixture of short to longalkanes is digested by the mutant microbe and compared to a similardigestion by the wild type halophile. The conditions of the digestionshould match the waterflood drive buffer or fluid. It should contain thesoluble carbon source such as molasses that will be used to supplementgrowth. The incubation should be long enough to measure degradation andloss of alkanes or a change in total hydrocarbon composition ifpetroleum oil is used. The measure of a good or preferred engineeredmicrobe is that it reduces viscosity. After digestion, there should be arelative increase in the lighter chain hydrocarbons and a reduction inthe heavier or higher molecular weight oil components. This shouldresult in a decrease in overall oil viscosity. The decrease in viscosityshould also be compared to the results from the wild type microbe thatstill has the ability to metabolize short chain alkanes.

Microbes that can cause a decrease in overall viscosity, and that canstill mobilize or dislodge oil as well as the wild type are then testedin an oil well field test. In a single well test, the microorganisms,along with nutrients similar to the culture collection medium used inExample 1, are pumped into a well that is at a salt level that isconducive for halophilic growth. The volume of liquid culture andnutrients should penetrate the reservoir for several meters beyond thewell bore. This could be several hundred gallons or more. Afterpenetration, the well is shut in with no more liquid pumped for twoweeks. After the shut in period, the well is reopened and the amount ofboth oil and water removed from the well are measured. A successful MEORmicroorganism will show an increase in the number of microbes, anincrease in the amount of oil produced and a measurable reduction in theviscosity of the oil produced. As a control experiment, a similar testshould be done by simple stimulation of indigenous microbes by justinjecting nutrients into the oil well. As another control, test, anothertest with chemical surfactants should be run on the same well or similarwell.

Example 3

In this example microbes are isolated for their ability to produceextracellular polymers in a high salt environment with little or noability to consume light weight hydrocarbons. One of the most importantfactors for waterflood is sweep efficiency. Permeability variation andfractures will cause high flow of fluid through some areas and little orno flow in other areas. Polymers are useful in oil recovery because theycan block the flow of fluid through the low resistance channels. Theyalso thicken the water to make a better fluid to drive the oil out ofthe reservoir. Both bio-polymers and chemical polymers can be used forthis purpose. Most chemical polymers are less effective at highsalinity. Bio-polymers, especially those produced by microbes, whichlive in high salinity environments, are more useful for oil recoveryfrom high salinity reservoirs. Bio-polymers are variable in theirviscosity in high salinity brine. Pfiffner et al. reported in Appl.Environ. Microbiol. 1986, 51:1224-1229 isolating over 200 bacterialstrains. The isolated strains can grow anaerobicly at 50° C. in up to10% NaCl and could produce various levels of extracellularpolysaccharides. The isolation media was Medium E also used by Jennemanet al. 1984, Soc. Pet. Eng. J. 24:33-37. The medium is a sucrose-mineralsalts medium with 5% (wt/vol.) NaCl. Environmental samples were from anumber of sources, most of which contented oil.

Unlike surfactant production, the genes required for extracellularpolymer production are not necessarily under the control of the samepromoter or clustered with the genes required for petroleum metabolism.However, most of the microorganisms used in MEOR have been isolated fromoil rich environments or are the indigenous bacteria that live in an oilreservoir where the only source on carbon is oil. Although Pfiffner andco-workers were able to maintain both growth and polymer production onsucrose it is not clear that these isolated strains had lost theirability to metabolize petroleum hydrocarbons. Therefore, their use aseither an inoculating microorganism or as an indigenous microbe couldlead to loss of short chain alkanes and a corresponding increase inviscosity.

To prevent this from occurring, the isolation procedure should include atest for the ability of isolate to metabolize short chain alkanes. Thiscan be done by microbiological techniques such as picking colonies thatcan grow on agar plates with a simple sugar carbon source, but thatcannot grow on plates with a short chain alkane as the only carbonsource. Alternatively, a radioactive or florescent probe to a gene thatis characteristic of alkane degradation such as those described inExamples 1 and 2 could be used to screen isolate. Isolated microbes thatgrow well on a simple carbon source and produce high levels of polymercould revert back to consuming light oil if insufficient sugar weresupplied.

To prevent polymer producing halophilic microorganisms from picking upgenes from indigenous halophiles in a naturally high salt reservoirother genes could be added. For example, the modified gene that codesfor the halophilic long chain alkane hydroxylase would give the polymerproducing microbes an additional carbon source.

Ideally the production of polymers should be controllable. Ifbio-polymers could be produced in the high flow channels and highporosity regions it would force the flow of fluid into the unswept areasthat were still retaining petroleum. Therefore having the genes thatcontrol the production of extracellular polymer under the control of aninducible promoter would be best. Certain levels of soluble carbonsources or metabolites may lead to increased polymer production.Identification of halophilic microorganisms that can only producepolymers when induced by certain metabolites such as xylose or arabinosewould provide a means for control of polymer production. The inductionof polymer production can be done after the reservoir is inoculated bystarved or sporilated halophiles. Once in place, especially in thehighest flow areas of the reservoir, the metabolite can be supplied bythe waterflood fluid. This is expected to cause the most growth andproduction of polymer by the microbes that had infiltrated into thehighest flow channels.

Therefore, the first step is selecting from many isolated halophilic themicrobes that had the ability to: produce salt tolerant polymers, formspores, produce polymer only when supplied a certain group of carbonsources, grow without consuming short chain alkanes. The second step isto make sure that the selected microbes will not pick up or turn on agene for short chain alkane consumption. The third step is to test themicrobes in a field test of oil producing wells and water injectionwells that is experiencing a problem with high flow zones causing thewater flood fluid to by-pass much of the residual oil in place.

Example 4

A DNA construct for heterologous expression in Haloferax volcanii wassynthesized that contained a modified version of the Haloferax volcaniiHMG-CoA reductase gene promoter (Nuttall, et al., Biochem. J, 2000), aterminator adapted from the H. volcanii DNA gyrB gene, and a modifiedversion of the Aequorea victoria Green Fluorescent Protein (GFP), knownas smRS-GFP, containing four mutations, including Ser65Thr, Phe99Ser,Met153Thr, Val163Ala (the Phe99Ser, Met153Thr, and Val163Alamodifications confer the “soluble modified” protein properties; theSer65Thr modification comprises the red-shifted mutation). The DNAconstruct was linearized by restriction digestion and ligated intopNG168, an archaeal/E. coli shuttle vector containing an archaeal originof replication and a mevinolin resistance gene for selection in archaea(S. DasSarma, 1995).

Haloferax volcanii strain DS2 (ATCC 29605) was transformed with thepNG168-smRS-GFP plasmid, or pNG168, using established PEG-basedtransformation methods (Dyall-Smith, The Halohandbook: Protocols forHaloarchaeal Genetics, 2009), and transformants were selected on richmedium plates containing 2 μg/ml lovastatin (Tocris). After four days'growth at 46° C., transformant colonies were picked and patched to richmedium plates containing lovastatin and were grown overnight at 46° C.

The presence of the smRS-GFP gene in transformant Haloferax volcaniistrains was confirmed by isolation of plasmid DNA from transformantHaloferax volcanii strains and DNA sequencing of the smRS-GFP gene.Transformant H. volcanii pNG168-smRS-GFP and pNG168 transformants wereviewed under fluorescence microscopy. H. volcanii pNG168-smRS-GFPtransformants exhibited strong fluorescence, demonstrating smRS-GFPexpression, whereas pNG168 transformants did not.

Example 5

In a specific example of testing a modified LadA gene, for proteinexpression by an obligate halophile, Haloferax volcanii, a small numberof lysine resides in the wide type LadA sequence were changed to codonsthat code for negatively charged amino acids. For expression of LadA,with an epitope tag comprised of six histidines (i.e., 6×His) at theprotein N-terminus, wild-type LadA DNA sequence (Geobacillusthermodenitrificans NG80-2) was synthesized and cloned into pET-28a(+)(Novagen, USA; DNA 2.0, Menlo Park, Calif.). The resulting expressionplasmid was transformed into BL21 E. coli (DE3) (New England Biolabs,Cat # C2527H) following the manufacturer's protocol, and thetransformant strain was named GFF40.

Three DNA constructs for heterologous expression in Haloferax volcaniiwere synthesized that contained a modified version of the Haloferaxvolcanii HMG-CoA reductase gene promoter (Nuttall, et al., Biochem. J,346(Pt 2) 251-254, 2000), a start codon followed by DNA sequencesencoding an epitope tag consisting of six histidine residues at theN-terminus of following protein coding sequences, three differentmodified versions of the Geobacillus thermodenitrificans NG80-2 LadAgene, and a synthetic terminator sequence t.Syn (Allers, et al., Appl.Environ. Microbiol., 76(6):1759-69, 2010). Of the three versions of theLadA gene, one version consisted of a DNA sequence codon optimized forexpression, in Haloferax volcanii, of the published LadA proteinsequence from Geobacillus thermodenitrificans. The two other versions ofthe LadA gene consisted of sequences identical to the first, except forchanges at codons that specified changes at 3 amino acid residues(K263E, K267E, and K276D) and 5 amino acid residues (K263E, K267E,K276D, K357D, and K358E) in the published LadA protein sequence. DNAsequences for the protein coding sequences and terminator for the threeDNA constructs, were synthesized (DNA 2.0, Menlo Park, Calif.) and PCRamplified using a reverse primer (prGFF32, SEQ ID NO: 12), matchingterminator DNA sequence, and a long upstream primer (prGFF36, SEQ ID NO:11), incorporating the entire HMG-CoA reductase promoter, to generatethe final constructs containing promoter, protein coding sequence, andterminator. The PCR product was linearized by restriction digestion, andligated into pNG168 (Allers T. and Mevarech M. Nature Reviews 6, 58-73,2005 and Supplement and AY291460.1), an archaeal/E. coli. shuttle vectorcontaining an archaeal origin of replication and a mevinolin resistancegene for selection in archaea (DasSarma, S., in Archaea: A LaboratoryManual (eds DasSarma, S. & Fleischmann, E. M.) 241-252 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1995), resulting inthe generation of plasmids pNG168-LADA-wt-optHv1 (SEQ ID NO: 5),pNG168-LADA-mut3-optHv1 (SEQ ID NO: 6), and pNG168-LADA-mut5-optHv1 (SEQID NO: 7) (indicating plasmids harboring DNA sequences encoding thewildtype LadA protein (SEQ ID NO: 8), the LadA version incorporatingthree amino acid substitutions (SEQ ID NO: 9), and the LadA versionincorporating five amino acid substitutions (SEQ ID NO: 10),respectively). DNA sequencing was carried out on resulting plasmids,verifying correct DNA sequence throughout DNA constructs and acrossligation junctions within the plasmids.

Haloferax volcanii strain DS2 (ATCC 29605) was transformed with thethree LadA expression plasmids, or with the original pNG168 plasmid,using established PEG-based transformation methods (Dyall-Smith, TheHalohandbook: Protocols for Haloarchaeal Genetics, 2009), andtransformants were selected on rich medium plates containing 2 μg/mllovastatin (Tocris). After four days' growth at 46° C., transformantcolonies were picked and transferred to rich medium plates containinglovastatin and were grown overnight at 46° C. Transformant strains werenamed GFF36, GFF31, and GFF22, for strains harboringpNG168-LADA-wt-optHv (Synthesized LadA with no mutated amino acids),pNG168-LADA-mut3-optHv1 (three mutated amino residues (K263E, K267E,K276D), and pNG168-LADA-mut5-optHv1 (five mutated amino residues (K₂₆₃E,K267E, K276D, K357D, K358E)), respectively.

Cloning, Expression, and Purification

Synthesized wild-type native LadA was cloned into pET-28a(+) (Novagen,USA) with a 6×His tag at the N terminus and expressed in E. coli BL21(DE3) (New England Biolabs, Cat # C2527H) following the manufacturer'sprotocol, and the clone expressed LadA named GFF40. GFF40 cells weregrown at 37° C. in 100 mL of LB medium containing 100 μg/ml kanamycin,to an OD600 of 0.6-0.8, and were then continuously induced with 0.5 mMisopropyl-β-D-thiogalactopyranoside (IPTG) at 37° C. for a further 3hours.

Transformations of Haloferax volcanii DS2 with mutated LadA were carriedout by mixing 10 μL 0.5M EDTA (pH8.0) with 100 μL Haloferax volcaniicompetent cells gently and incubating for 10 minutes at roomtemperature. 1 μg pNG168 plasmid constructs was added to the cells/EDTA,gently mixed and incubated at room temperature for 5 minutes, followedby adding 110 μL 60% PEG (v/v) in unbuffered spheroplast solution (2 MNaCl, 17 mM KCl, 15% (w/v) sucrose], and incubated at room temperaturefor 30 minutes. Then 600 μL HV-YPC liquid medium was added to the cellsolution, cells were recovered at 65,000 rpm for 5 minutes, andresuspended in 600 μL HV-YPC, incubated at 37° C. for 2 hours, platingon HV-YPC-agar plates, incubated the plates at 47° C. for 3 days. A6×His-tag was added to the N-terminal of LadA of the LadA genes cloned.Haloferax volcanii (HV) transformant GFF31 (LadA hv3mut) controlled byover-expression promoter hv-HMG-CoA, Haloferax volcanii transformed withempty vector pNG168 (HVev), and Haloferax volcanii were grown at 47° C.for 20 hours. Cells were harvested by centrifugation at 4200 rpm for 10min at room temperature. E. coli cells were resuspended in lysis bufferA [40 mM Tris-HCl (pH 8.0) and 10 mM NaCl], Haloferax volcanii wereresuspended in lysis buffer B [40 mM Tris-HCl (pH 8.0) and 2 M NaCl],and disrupted by sonication, following centrifugation for 2 minutes at14,000 g. The soluble cell lysate was applied onto a Ni2+-chelatingaffinity column (100 μL preequilibrated with lysis buffer). Thecontaminant protein was washed off with 1000 μL wash buffer [lysisbuffer and 20 mM imidazole], and the LadA was eluted with 100 μL elutionbuffer [lysis buffer and 200 mM imidazole]. Eluted LadA from E. coli andHaloferax volcanii transformants were confirmed by gel electrophoresisfollowed by His-tag in-gel stain and coomassie blue stain.

The results are shown in FIG. 3, which is a photograph of a gel showingthe expression of ladA in E. coli and in Haloferax volcanii. The Novagen(now part of EMD) pET-28a(+) vector carries an N terminal 6His Tag,thrombin cleavage site (6 residues: Leu-Val-Pro-Arg-Gly-Ser), T7 Tag (11residues, used for express studies using anti-T7 Tagantibody) and a fewresidues between these site/Tags, followed by multi-cloning site, thusgive ˜34 to 45 amino acids to the N-terminal of the expressed protein.For mutated LadA, the one we purified from HV has 7 residues added toits N-terminal (MHHHHHH-LadA).

Enzyme Activity Assays

Partially purified LadA E. coli and Haloferax volcanii transformantswere assayed for alkane monooxygenase activity following the methoddescribed by Feng et al. (2007) with modification. The reaction mixturescontained 50 mM Tris-HCl (pH 7.5), 1 mM hexadecane, 1 mM each of FMNH2and MgSO4, and variable amounts of each purified protein. A mixturewithout enzyme, a mixture with approximately equal amounts of elutedprotein from Haloferax volcanii, a mixture with approximately equalamounts of eluted protein Haloferax volcanii transformant with emptyvector pNG168, were used as controls. The mixtures were incubated at 60°C. for up to one hour before extraction with hexane. Aliquots of thehexane extracted long chain hydrocarbon substrate were analyzed by highperformance gas chromatography. The hexadecane was eluted from a AgilentTechnologies High performance capillary column 19091J-413 HP-5(crosslinked 5% PHME siloxane) 30 meter 0.32 mm column. The gaschromatograph was a Hewlett Packard 5890 series II with a flameIonization detector. The carrier gas was helium with a flow rate ofabout 15 ml per minute and a starting temperature of 120° C. whichincreased at the rate of 15° C. per minute until the final temperatureof 280° C. was reached. Quantization and the determination of activitywas done by comparison to injection and elution of hexadecane standardsolutions and control digestions without added proteins. This method wasused to determine if the modified proteins have equivalent activity tothe wide type LadA and also to determine if the halo-adapted enzyme wasactive at higher salt concentrations than the wild type enzyme.

1. A method of enhancing oil recovery comprising introducing into an oilreservoir a microorganism capable of growing in an environment of highsalinity, which microorganism is deficient in its ability to degradeshort chain hydrocarbons of about 12 carbons or less.
 2. The method ofclaim 1 wherein the oil reservoir is selected from the group consistingof underground reservoirs, producing wells, non-producing wells,experimental wells, exploratory wells, oil sands and other sources ofheavy oil.
 3. The method of claim 1 wherein the growth of saidmicroorganism is obligately dependent on high salinity.
 4. The method ofclaim 3 wherein the microorganism is an archaeon or a bacterium.
 5. Themethod of claim 3 wherein the microorganism is present in a culture ofmicroorganisms comprising a plurality of microorganisms that areobligatory halophiles and are deficient in their ability to degradeshort chain hydrocarbons of about 12 carbons or less, wherein the growthof said culture is obligately dependent on high salinity.
 6. The methodof claim 5 wherein said obligatory halophile microorganisms aredeficient in their ability to degrade short chain hydrocarbons of lessthan about 20 carbons.
 7. The method of claim 3 wherein saidmicroorganism is able to grow in a salinity of about 5% or higher. 8.The method of claim 3 wherein said microorganism is inhibited inacquiring the ability to grow at salinity below about 5% frommicroorganisms indigenous or contaminating said reservoir.
 9. The methodof claim 3 wherein in said microorganism one or more metabolic pathwaysdegrading short chain hydrocarbons of about 12 carbons or less are downregulated, mutated or deleted.
 10. The method of claim 3 wherein saidmicroorganism naturally lacks the ability to degrade short chainhydrocarbons of about 12 carbons or less.
 11. The method of claim 3wherein said microorganism has the ability to utilize aromatichydrocarbons.
 12. The method of claim 3 wherein said microorganism hasthe ability to utilize hydrocarbon chains of greater than about 12carbons.
 13. The method of claim 3 wherein said microorganism has theability to utilize modified hydrocarbons containing sulfur.
 14. Themethod of claim 3 wherein said microorganism has the ability to utilizemodified hydrocarbons containing nitrogen.
 15. The method of claim 3wherein said microorganism has the ability to utilize simple carbonsselected from the group comprising glucose, sucrose, mannose, starch,glycerin, organic acids, and other simple sugars.
 16. The method ofclaim 3 wherein said microorganism has the ability to producesurfactants.
 17. The method of claim 3 wherein said microorganism hasthe ability to produce extra cellular polymers.
 18. The method of claim3 wherein said microorganism (i) contains functional genes for themetabolism of high molecular weight hydrocarbons; (ii) lacks functionalgenes for the transport and oxidation of short chain alkanes at the cellmembrane; (iii) contains functional genes for the production ofsurfactants; and (iv) is regulated to express said functional genes andgrow in a high salt environment within said reservoir.
 19. The method ofclaim 3 further comprising the step of injecting a nutrient mixture intosaid reservoir.
 20. The method of 3 further comprising the step ofwater-flooding said reservoir with low salinity fluid or a fluidcontaining a compound toxic to said microorganism to reduce theconcentration of halophilic microorganisms that have the ability toutilize short chain hydrocarbons of about 12 carbons or less.
 21. Amicroorganism that (i) is a halophile, and (ii) is deficient in itsability to degrade short chain hydrocarbons of about 12 carbons or less.22. The microorganism of claim 21 which is an obligatory halophile. 23.The microorganism of claim 22 that is an archaeon or a bacterium. 24.The microorganism of claim 23 which is an archaeon.
 25. Themicroorganism of claim 21 that naturally has at least one of properties(i) and (ii).
 26. The microorganism of claim 21 that is engineered tohave at least one of properties (i) and (ii).
 27. The microorganism ofclaim 21 which is able to grow in a salinity of about 5% or higher. 28.The microorganism of 21 which is additionally inhibited in acquiring theability to grow at salinity below about 5% from microorganismsindigenous in or contaminating an oil reservoir.
 29. The microorganismof claim 21 which is additionally deficient in its ability to degradehydrocarbons of about 20 carbons or less.
 30. The microorganism of claim21 which additionally has the ability to utilize aromatic hydrocarbons.31. The microorganism of claim 21 which additionally has the ability toutilize hydrocarbon chains of greater than 12 carbons.
 32. Themicroorganism of claim 21 which additionally has the ability to utilizehydrocarbons containing sulfur.
 33. The microorganism of claim 21 whichadditionally has the ability to utilize modified hydrocarbons containingnitrogen.
 34. The microorganism of claim 21 which additionally has theability to utilize simple carbons from the group comprising; glucose,sucrose, mannose, starch, glycerin, organic acids, and other simplesugars.
 35. The microorganism of claim 21 which additionally has theability to produce surfactants.
 36. The microorganism of claim 21 whichadditionally has the ability to produce extra cellular polymers.
 37. Themicroorganism of claim 21 which (i) contains functional genes for themetabolism of high molecular weight hydrocarbons; (ii) lacks functionalgenes for the transport and oxidation of short chain alkanes at the cellmembrane; (iii) contains functional genes for the production ofsurfactants; and (iv) is regulated to express said functional genes andgrow in a high salt environment within an oil reservoir.
 38. A cultureor consortium comprising a microorganism of claim 21 or claim
 37. 39. Aculture or consortium consisting essentially of microorganisms accordingto claim 21 or claim
 37. 40. A culture or consortium consisting ofmicroorganisms according to claim 21 or 37.